Antithrombotic n-amidinopiperidine and benzamidine derivatives

ABSTRACT

Compounds of formula (I), pharmaceutically acceptable salts thereof, and pharmaceutically acceptable solvates of either entity, wherein A is optionally monosaturated C 1  -C 4  alkylene optionaly monounsaturatred with C 1  -C 4  alkyl; B is C 1  -C 3  alkylene optionally substituted with C 1  -C 4  alkyl; R 1  is N-amidino-4-piperidyl or 4-amidinophenyl; R 2  is C 4  -C 12  alkyl; (C 3  -C 8  cycalkyl)C 1  -C 4  alkylene; optionally methylene-bridged C 5  -C 8  cycloalkyl optionally substituted with one to three C 1  -C 4  alkyl groups or with hydroxy; C 5  -C 8  alkenyl; C 5  -C 8  cycloalkenyl optionally subsituted with C 1  -C 4  alkyl; piperidyl N-substituted with C 1  -C 4  alkyl; tetrahydrothiopyranyl or tetrahydropyranyl; and R 3  is H or C 1  -C 4  alkyl optionally substituted with C 1  -C 4  alkoxy or with hydroxy; or R 2  and R 3 , together with the nitrogen atom to which they are attached, form a piperidine ring which is optionally substituted wiht C 1  -C 4  alkyl or is optionally benzo-fused; are potent and selective thrombin inhibitors useful in the treatment of, inter alia, deep vein thrombosis; reocclusion following thrombolytic therapy; chronic arterial obstruction; peripheral vascular disease; acute myocardial infarction; unstable angina; atrial fibrillation; thrombotic stroke; transient ischaemic attacks; restenosis and occlusion following angiopasty; or neurodegenerative disorders.

This application was filed under 35 U.S.C. §371 based on PCT/EP96/04610,which was filed on Oct. 21, 1996 which claims priority from GreatBritain application No. 9522495.2 which was filed on Nov. 2, 1998 and isnow abandoned.

This invention relates to a series of N-amidinopiperidine andbenzamidine derivatives, which are antithrombotic agents, having utilityin a variety of therapeutic areas including the prevention and/ortreatment of deep vein thrombosis (DVT) after surgery, major medicalillness, paralysis, malignancy, prolonged immobilisation trauma,application of lower limb plaster casts, or fractures of the lower limbsor pelvis; recurrent DVT; DVT during pregnancy when there is a previoushistory thereof; reocclusion following thrombolytic therapy; chronicarterial obstruction; peripheral vascular disease; acute myocardialinfarction; unstable angina; atrial fibrillation; thrombotic stroke;transient ischaemic attacks; disseminated intravascular coagulation;coagulation in extra-corporeal circuits; occlusion of arterio-venousshunts and blood vessel grafts (including coronary artery by-passgrafts); and restenosis and occlusion following angioplasty. They alsohave utility as an adjunct to thrombolytic therapy.

The compounds of the invention are potent and selective inhibitors ofthrombin, which is the final serine protease enzyme in the coagulationcascade. The prime function of thrombin is the cleavage of fibrinogen toproduce fibrin which forms linear insoluble polymers which, in turn, arecross-linked by factor XIIIa, itself activated by thrombin. In addition,thrombin regulates its own production by activation of factors V andVIII earlier in the cascade. It also has important actions at thecellular level, where it acts on specific receptors to cause plateletaggregation, endothelial cell activation and fibroblast proliferation.Thus thrombin has a central regulatory role in haemostasis and thrombusformation.

Clearly then, potent, selective and orally bioavailable thrombininhibitors represent an attractive target for the convenient therapeuticcontrol of thrombosis. In addition, thrombin potently causes neuriteretraction and therefore a thrombin inhibitor is of potentialtherapeutic utility in the treatment of acute and chronicneurodegenerative disorders. Furthermore, the compounds disclosed hereinare of potential value in the treatment of inflammatory disorders andscarring, and in wound healing.

Because of their potential as substrate mimics, arginine derivativeshave been explored as thrombin inhibitors and this work led to thediscovery of argatroban (see Cardiovascular Drug Rev., 1991, 9, 247). Inturn, other research groups have sought to express the basic argininefunction in alternative structures; for example, WO-A-95/13274 disclosesamidinophenylalanine and amidinopyridylalanine derivatives asantithrombotic agents. Further variations on the theme of argininemimicry amongst thrombin inhibitors are represented by, inter alia, theguanidinyl- and amidinyl-substituted heterocyclic compounds disclosed inEP-A-0623595.

The compounds of the present invention are potent thrombin inhibitors,selective (in comparison with their inhibition of, for example, trypsin,plasmin, butyrylcholinesterase and elastase), well tolerated and orallybioavailable. Accordingly, the present invention provides a compound offormula (I): ##STR1## pharmaceutically acceptable salts thereof, andpharmaceutically acceptable solvates of either entity,

wherein A is optionally monounsaturated C₄ -C₅ alkylene optionallysubstituted with C₁ -C₄ alkyl;

B is C₁ -C₃ alkylene optionally substituted with C₁ -C₄ alkyl;

R¹ is N-amidino-4-piperidyl or 4-amidinophenyl;

R² is C₄ -C₁₂ alkyl; (C₃ -C₈ cycloalkyl)C₁ -C₄ alkylene; optionallymethylene-bridged C₅ -C₈ cycloalkyl optionally substituted with one tothree C₁ -C₄ alkyl groups or with hydroxy; C₅ -C₈ alkenyl;

C₅ -C₈ cycloalkenyl optionally substituted with C₁ -C₄ alkyl; piperidylN-substituted with C₁ -C₄ alkyl; tetrahydrothiopyranyl ortetrahydropyranyl;

and R³ is H or C₁ -C₄ alkyl optionally substituted with C₁ -C₄ alkoxy orwith hydroxy;

or R² and R³, together with the nitrogen atom to which they areattached, form a piperidine ring which is optionally substituted with C₁-C₄ alkyl or is optionally benzo-fused.

In the above definition, unless otherwise indicated, alkyl and alkoxygroups having three or more carbon atoms and alkenyl groups having fouror more carbon atoms may be straight-chain or branched-chain.

The compounds of formula (i) contain two or more asymmetric centres andthus can exist as stereoisomers, i.e. as enantiomers or asdiastereoisomers as well as mixtures thereof.

The preferred stereoisomers are of formula (IA): ##STR2##

Also included in the invention are radiolabelled derivatives ofcompounds of formula (I) which are suitable for biological studies.

The pharmaceutically acceptable salts of the compounds of formula (I)are, for example, non-toxic acid addition salts formed with inorganicacids such as hydrochloric, hydrobromic, sulphuric and phosphoric acid,with organo-carboxylic acids, or with organo-sulphonic acids. Compoundsof formula (I) can also provide pharmaceutically acceptable metal salts,in particular non-toxic alkali metal salts, with bases. Examples includethe sodium and potassium salts.

A preferred group of compounds of formula (I) is that wherein A isbutylene; B is C₁ -C₂ alkylene; R² is C₄ -C₆ alkyl; (C₃ -C₆cycloalkyl)CH₂ ; C₅ -C₈ cycloalkyl optionally substituted with one tothree methyl groups or with hydroxy; norbornyl; C₆ -C₇ cycloalkenyloptionally substituted with methyl; piperidyl N-substituted with methyl;tetrahydrothiopyranyl or tetrahydropyranyl; R³ is H, methyl or ethyl; orR² and R³, together with the nitrogen atom to which they are attached,form a piperidine ring substituted with methyl; and R¹ is as previouslydefined for formula (I).

A more preferred group of compounds of formula (i) is that wherein A isbutylene; B is methylene; R¹ is N-amidino-4-piperidyl; R² is 3-pentyl;(C₅ -C₆ cycloalkyl)CH₂ ; cyclopentyl; cyclohexyl optionally substitutedwith one or two methyl groups; 2-hydroxycyclohexyl; 2-norbornyl;cycloheptyl; cyclooctyl; cyclohexenyl optionally substituted withmethyl; cycloheptenyl; 3-tetrahydrothiopyranyl; 4-tetrahydrothiopyranylor 3-tetrahydropyranyl; and R³ is H, methyl or ethyl.

A most preferred group of compounds of formula (I) is that wherein A isbutylene; B is methylene; R¹ is N-amidino-4-piperidyl; R² is cyclohexyl;3-methylcyclohexyl; 3,3-dimethylcyclohexyl; 3,5-dimethylcyclohexyl;2-norbornyl; cycloheptyl; cyclooctyl; 3-cyclohexenyl;3-methyl-3-cyclohexenyl or 4-cycloheptenyl; and R³ is H or methyl.

Particularly preferred individual compounds of the invention include:

N-(N-cycloheptyl-N-methyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine;

N-(N-4-cycloheptenyl-N-methyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine;

N-(N-methyl-N-3(R)-methylcyclohexyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine; and

N-(N-cyclohexyl-N-methyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine;

and pharmaceutically acceptable salts thereof, and pharmaceuticallyacceptable solvates of either entity.

In another aspect, the present invention provides processes for thepreparation of compounds of formula (I), their pharmaceuticallyacceptable salts, and pharmaceutically acceptable solvates of eitherentity.

A compound of formula (I) may be prepared from a compound of formula(II): ##STR3## wherein R⁴ is C₁ -C₄ alkyl or benzyl, and A, B, R¹, R²and R³ are as previously defined for formula (I). Depending on thenature of R⁴, it may be removed by conventional acid- or base-catalysedhydrolysis, protonolysis or hydrogenolysis. In cases where the amidinogroup in R¹ is also protected, it is particularly convenient to removethe amidine protecting group (P¹) in the same reaction step as theremoval of R⁴ ; P¹ may be a typical amine protecting group such ast-butoxycarbonyl (Boc) or benzyloxycarbonyl (Cbz).

A preferred method of preparing a compound of formula (I) wherein R¹ isN-amidino-4-piperidyl is by single step deprotection of a compound offormula (IIA): ##STR4## wherein R⁴ and P¹ are removable under the samereaction conditions. Preferably R⁴ is t-butyl, P¹ is Boc and thereaction is effected by protonolysis using, for example, hydrogenchloride or trifluoroacetic acid at from about 0° C. to about roomtemperature. Typically, a solution of a compound of formula (IIA) in asuitable solvent such as dichloromethane is saturated with hydrogenchloride at about 0° C. and then allowed to warm to room temperature.Full deprotection is normally complete within 2 to 6 hours at roomtemperature.

Alternatively, a 1:1 mixture of trifluoroacetic acid and a suitablesolvent such as dichloromethane may be employed as the reaction mediumat from about 0° C. to about room temperature.

A preferred method of preparing a compound of formula (I) wherein R¹ is4-amidinophenyl is by hydrolysis of the corresponding ester of formula(IIB): ##STR5## wherein R⁴ is typically ethyl and/or benzyl. Thereaction may be acid- or base-catalysed, but is generally carried outusing an alkali metal hydroxide such as sodium or potassium hydroxide inaqueous solution, optionally in the presence of a suitable cosolvent, atfrom about room temperature to about 100° C. Preferred conditions arethe use of aqueous sodium hydroxide solution, with 1,4-dioxan ascosolvent, at about room temperature.

Thus novel intermediates of formula (II) wherein the amidino group in R¹is optionally protected and A, B, R¹, R², R³ and R⁴ are as previouslydefined for formula (II) also form part of the invention.

A compound of formula (IIA) may be prepared from a compound of formula(III): ##STR6## wherein A, B, R⁴ and P¹ are as previously defined forformula (II), by conventional alkylation of the primary amino group.When R³ is not H, two separate N-alkylation steps are required,generally with R² being introduced first. Only when R² and R³ areidentical, i.e. C₄ alkyl, may the two alkylations being carried out in asingle step. Similarly, when R³ and the piperidyl N-substituent of R²are identical, i.e. C₁ -C₄ alkyl, these may be introduced in a singlestep. When NR² R³ forms a piperidine ring; the required bis-alkylationis preferably effected in a single step.

Of the plethora of N-alkylation procedures available, one example of ageneral reaction which may be employed is that of a compound of formula(III) with a compound of formula R² X, wherein R² is as previouslydefined for formula (II), and X is a suitable leaving group, e.g. halo(preferably chloro, bromo or iodo), C₁ -C₄ alkanesulphonyloxy,trifluoromethanesulphonyloxy or aryisulphonyloxy (preferablybenzenesulphonyloxy or p-toluenesulphonyloxy), in the presence of anappropriate base, e.g. the carbonate or bicarbonate salt of an alkali oralkaline earth metal, or a tertiary amine, in a suitable solvent such asa C₁ -C₄ alkanol, 1,2-dimethoxyethane, acetonitrile, dimethylformamideor N,N-dimethylacetamide, optionally in the presence of sodium orpotassium iodide. The reaction may be conducted at from about 0° C. toabout 150° C., preferably at from about room temperature to about 85° C.

If appropriate, a conventional amine protecting group strategy may beused for the introduction of R².

R³, when not H, may then be similarly introduced. When R³ is C₂ -C₄alkyl substituted with hydroxy, it may be convenient to react a compoundof formula (III) with an appropriate epoxide-containing R³ precursor,optionally in the presence of a tertiary amine base, e.g. triethylamine,and preferably in a suitable solvent such as a C₁ -C₄ alkanol. Thereaction can be conducted at from about 0° C. to about 150° C.,preferably at from room temperature to about 60° C. using methanol assolvent. When R³ is 2-hydroxyethyl, an ethylene oxide equivalents ispreferably employed. Thus a compound of formula (III) may be reactedwith ethylene carbonate in a suitable solvent such as dimethylformamideat about 120° C.

Alternatively, as a further example of a general N-alkylation reaction,the desired transformation may be achieved by reductive alkylation of acompound of formula (III) using the appropriate aldehyde- orketone-containing R² precursor. The substrate of formula (III) andcarbonyl reagent may be reacted together under conventional catalytichydrogenation conditions or in the presence of sodium cyanoborohydrideor sodium triacetoxyborohydride, in a suitable solvent such as methanol,ethanol, dichloromethane, 1,2-dichloroethane or tetrahydrofuran,optionally in the presence of glacial acetic acid or in the presence ofpre-activated molecular sieves, at about room temperature.

Preferably, a compound of formula (III) is treated initially with from1.1 to 2.0 molecular equivalents of the appropriate aldehyde or ketone,optionally in the presence of from 1.0 to 2.1 molecular equivalents ofglacial acetic acid or in the presence of pre-activated 4A molecularsieves, in tetrahydrofuran or 1,2-dichloroethane, at about roomtemperature followed, about 5 minutes later, with from 1.4 to 2.0molecular equivalents of sodium triacetoxyborohydride.

R³, when not H, may be similarly introduced subsequently, although up toabout 3 molecular equivalents of the appropriate aldehyde or ketone andof sodium triacetoxyborohydride may be required to effect a reasonableconversion. When R³ is methyl, it is particularly convenient to useabout 4 molecular equivalents of aqueous formaldehyde solution and about2 molecular equivalents of sodium triacetoxyborohydride indichloromethane at about room temperature. When both R³ and thepiperidyl N-substituent of R² are methyl, about 5 molecular equivalentsof aqueous formaldehyde solution may be more desirable. When NR² R³forms a piperidine ring, a compound of formula (III) may be treatedinitially with about 1.5 molecular equivalents of the appropriateglutaraldehyde followed, about 0.5 hour later, with about 1 molecularequivalent of sodium triacetoxyborohydride at about room temperature.

A compound of formula (III) may be prepared from a compound of formula(IV): ##STR7## wherein P², like P¹ is a typical amine protecting groupand A, B, R⁴ and P¹ are as previously defined for formula (III), withthe proviso that P² is selectively removable in the presence of P and R⁴; preferably P² is 9-fluorenylmethoxycarbonyl (Fmoc). The particularprotecting group can be removed under standard conditions which, in thecase of Fmoc, are treatment with about a 10-fold excess of piperidine ina suitable solvent such as tetrahydrofuran at about room temperature.

A compound of formula (IV) may be prepared by coupling a compound offormula (V): ##STR8## wherein A and P¹ are as previously defined forformula (IV), with a compound of formula (VI): ##STR9## wherein B, R⁴and P² are as previously defined for formula (IV). The coupling reactionmay be achieved using conventional amide bond-forming techniques, inparticular any one of a host of amino acid coupling variations. Forexample, the acid of formula (VI) may be activated using a carbodiimidesuch as 1,3-dicyclohexylcarbodiimide or1-ethyl-3-(3-dimethylamino-1-propyl)carbodiimide optionally in thepresence of 1-hydroxybenzotriazole and/or a catalyst such as4-dimethylaminopyridine, or by using a halotrisaminophosphonium saltsuch as bromotris(pyrrolidino)phosphonium hexafluorophosphate. Eithertype of coupling is conducted in a suitable solvent such asdichloromethane, optionally in the presence of a tertiary amine such asN-methyimorpholine or N-ethyidiisopropylamine (for example when eitherthe compound of formula (V) or the activating reagent is presented inthe form of an acid addition salt), at about 0° C. Preferably, from 1.1to 2.0 molecular equivalents of the activating reagent and from 2.0 to3.0 molecular equivalents of any tertiary amine present are employed.

A compound of formula (V) may be prepared from a compound of formula(VII): ##STR10## wherein P³, like P¹ and P², is a typical amineprotecting group and A and P¹ are as previously defined for formula (V),with the proviso that P³ is selectively removable in the presence of P¹; preferably P³ is benzyloxycarbonyl (Cbz). The particular protectinggroup can be removed under standard conditions which, in the case ofCbz, are hydrogenolysis in the presence of an appropriate catalyst in asuitable solvent. Preferably the deprotection is effected using apalladium on charcoal catalyst and ethanol as solvent.

A compound of formula (VII) may be prepared from a compound of formula(VIII): ##STR11## wherein A and P³ are as previously defined for formula(VII), by reaction with a S-alkylisothiourea derivative of formula (IX):##STR12## wherein R⁵ is C₁ -C₃ alkyl and P is as previously defined forformula (VII), in a suitable solvent such as dichloromethane, optionallyin the presence of a mercury(II) salt and a tertiary amine, at fromabout 0° C. to about 40° C. Preferably, R⁵ is methyl, a 10% excess ofthe compound of formula (IX) is used, and the reaction is carried out inthe presence of I molecular equivalent of mercuric chloride and a 2-foldexcess of triethylamine.

A compound of formula (VIII) may be prepared from a compound of formula(X): ##STR13## wherein P⁴, like P¹, P² and P³, is a typical amineprotecting group and A and P³ are as previously defined for formula(VIII), with the proviso that P⁴ is selectively removable in thepresence of P³ ; preferably P⁴ is t-butoxycarbonyl (Boc). The particularprotecting group can be removed under standard conditions which, in thecase of Boc, are as described previously for the conversion of acompound of formula (IIA) to a compound of formula (I).

A compound of formula (X) may be prepared by reaction of a compound offormula (XI): ##STR14## wherein X is a suitable leaving group aspreviously defined and A and P³ are as previously defined for formula(X), with the anion of a compound of formula (XII): ##STR15## wherein P⁴is as previously defined for formula (X), under conditions similar tothose described previously for the alkylation of a compound of formula(III) with R² X. Preferably, X is methanesulphonyloxy, the anion of acompound of formula (XII) is generated using sodium hydride, the solventis dimethylformamide and the O-alkylation is conducted at about roomtemperature.

A compound of formula (XI) may be prepared from a compound of formula(XIII): ##STR16## wherein A and P³ are as previously defined for formula(XI), by standard procedures. For example, when X ismethanesulphonyloxy, by treatment of a compound of formula (XIII) withabout 2 molecular equivalents of methanesulphonyl chloride in thepresence of about 2 molecular equivalents of a tertiary amine, e.g.triethylamine, in a suitable solvent such as dichioromethane at fromabout 0° C. to about 17° C.

A compound of formula (XIII) may also be prepared by standard proceduresfrom a compound of formula (XIV): ##STR17## wherein A is as previouslydefined for formula (XIII). For example, when P³ is Cbz, by treatment ofa compound of formula (XIV) with about a 5% excess ofN-(benzyloxycarbonyloxy)succinimide in the presence of about a 10%excess of a tertiary amine, e.g. triethylamine, in a suitable solventsuch as dichioromethane at from about 0° C. to about room temperature.

The preferred S-enantiomer of a compound of formula (XIV) may beobtained therefrom by classical resolution procedures (see, for example,Rec. Trav. chim., 1971, 90 755) or by chromatographic resolutionprocedures using either a chiral auxiliary or a chiral stationary phase.Alternatively, de novo asymmetric synthetic methodology may be adopted.

A compound of formula (IIB) may be prepared from a compound of formula(XV): ##STR18## wherein A, B, R², R³ and R⁴ are as previously definedfor formula (II); preferably R⁴ is benzyl. This may be achieved byconversion of the nitrile group to the required amidine via anintermediate imino ether under conventional conditions. For example, asolution of a compound of formula (XV) in a lower alkanol such asethanol is saturated with hydrogen chloride at about 0° C. to generatethe imino ether hydrochloride which, in turn, is treated as a solutionin the same alcohol with excess ammonia at about 50° C. followed byheating of the resulting mixture under reflux. The amidine may beconveniently isolated as an acid addition salt, e.g. hydrochloride, ifnecessary.

Alternatively, the imino ether free base may be generated in situ bytreatment of the nitrile with the appropriate alkali metal alkoxide inthe corresponding lower alkanol as solvent at about room temperature;for example, sodium ethoxide in ethanol. This step is followed bytreatment of the imino ether solution with a suitable ammonium salt,e.g. ammonium chloride, at about room temperature.

A compound of formula (XV) may be prepared from a compound of formula(XVI): ##STR19## wherein A, B and R⁴ are as previously defined forformula (XV), by analogy with the methods described previously for theconversion of a compound of formula (III) to a compound of formula(IIA).

A compound of formula (XVI) may be prepared from a compound of formula(XVII): ##STR20## wherein P⁵ is a typical amine protecting group and A,B and R⁴ are as previously defined for formula (XVI), with the provisothat P⁵ is selectively removable in the presence of R⁴ ; preferably P⁴is t-butoxycarbonyl (Boc). The particular protecting group can beremoved under standard conditions which, in the case of Boc, are asdescribed previously for the conversion of a compound of formula (IIA)to a compound of formula (I), but with the modification that thedeprotection is conducted in the presence of about 2 molecularequivalents of anisole. Typically, the reaction is carried out usingexcess trifluoroacetic acid as reagent and dichloromethane as solvent atabout 0° C.

A compound of formula (XVII) may be prepared by coupling a compound offormula (XVIII): ##STR21## wherein A is as previously defined forformula (XVII), with a compound of formula (XIX): ##STR22## wherein B,R⁴ and P⁵ are as previously defined for formula (XVII), by analogy withthe methods described previously for the conversion of compounds offormulae (V) and (VI) to a compound of formula (IV).

A compound of formula (XVIII) may be prepared from a compound of formula(XX): ##STR23## wherein P⁶ is a typical amine protecting group and A isas previously defined for formula (XVIII); preferably P⁶ is Boc. Theparticular protecting group can be removed under standard conditionswhich, in the case of Boc, are as described previously for theconversion of a compound of formula (XVII) to a compound of formula(XVI).

A compound of formula (XX) may be prepared from a compound of formula(XXI): ##STR24## wherein A and P⁶ are as previously defined for formula(XX), and 4-cyanophenol by analogy with the methods described previouslyfor the conversion of a compound of formula (XIII) to a compound offormula (X).

However, a more convenient strategy is to exploit the Mitsunobu reactionwhereby a compound of formula (XXI) and 4-cyanophenol can be coupleddirectly. Typically, about a 10% excess of a di(C₁ -C₃ alkyl)azodicarboxylate is added to a mixture of the two components, preferablywith the phenol also in about a 10% excess, in a suitable solvent suchas dichloromethane at about 0° C., followed by removal of the coolingbath.

A compound of formula (XXI) may be prepared from a compound of formula(XIV) as described previously for the conversion of a compound offormula (XIV) to a compound of formula (XIII). For example, when P⁶ isBoc, by treatment of a compound of formula (XIV) with di-t-butyldicarbonate in a suitable solvent such as ethyl acetate at from about 0°C. to about room temperature.

The intermediates required for the introduction of R² and R³ (when notH), when neither commercially available nor subsequently described, canbe obtained either by analogy with the processes described in thePreparations section or by conventional synthetic procedures, inaccordance with standard textbooks on organic chemistry or literatureprecedent, from readily accessible starting materials using appropriatereagents and reaction conditions.

Moreover, persons skilled in the art will be aware of variations of, andalternatives to, those processes described hereinafter in the Examplesand Preparations sections which allow the compounds defined by formula(I) to be obtained.

The pharmaceutically acceptable acid addition salts of the compounds offormula (I) may also be prepared in a conventional manner. For example asolution of the free base is treated with the appropriate acid, eitherneat or in a suitable solvent, and the resulting salt isolated either byfiltration or by evaporation under reduced pressure of the reactionsolvent. Pharmaceutically acceptable base addition salts can be obtainedin an analogous manner by treating a solution of a compound of formula(I) with the appropriate base. Both types of salt may be formed orinterconverted using ion-exchange resin techniques.

The biological activities of the compounds of the present invention weredetermined by the following test methods.

Chromogenic Assays

The inhibition of thrombin, trypsin, plasmin or factor Xa is measured in96 well plate chromogenic assays. The percentage inhibition and IC₅₀ arecalculated from triplicate samples of an 8 concentration dose-responsecurve. From the substrate Km and IC₅₀, the Ki for each inhibitor iscalculated. All assays are carried out in a total incubation of 200 μlof 50 mM HEPES and 150 mM NaCl at pH 8.0, and all compound dilutions arepreincubated with enzyme at room temperature for 15 minutes prior toaddition of substrate. After 30 minutes incubation at 30° C., the O.D.is measured at 405 nM in a 96 well plate reader. Thrombin activity ismeasured using bovine thrombin and S2238 (H-D-Phe-Pip-Arg-pNA), bovinepancreatic trypsin is assayed with S2222 (Benz-Isoleu-Glu-Gly-Arg-pNA),bovine plasma plasmin is assayed with Chromozym PL(Tosyl-Giy-Pro-Lys-pNA) and bovine factor Xa is assayed in 50 mM Tris,150 mM NaCl, pH 7.5 buffer with S2222.

Clotting Assays

Thrombin time (TT) and activated partial thromboplastin time (APTT) aremeasured using Instrumentation Laboratories (IL) Test TT reagent and ILTest APTT (ellagic acid) reagent respectively in an AutomatedCoagulation Laboratory (ACL), according to the manufacturer'sinstructions.

In Vitro

To 1 ml aliquots of rat pooled plasma (citrated), a 1/100 volume of arange of compound concentrations is added and the resulting mixturespreincubated at room temperature for 15 minutes, after which the TT andAPTT are measured.

Ex Vivo

Compounds are dosed per os, intravenously or intraduodenally to rats.Pre- and post-dose blood samples are taken into citrate solution andplasma prepared. TT and APTT are measured as for in vitro assays.

In therapy, the compounds of formula (I), their pharmaceuticallyacceptable salts, and pharmaceutically acceptable solvates of eitherentity, can be administered alone, but will generally be administered inadmixture with a pharmaceutical carrier selected with regard to theintended route of administration and standard pharmaceutical practice.Preferably, they are administered orally in the form of tabletscontaining such excipients as starch or lactose, or in capsules orovules either alone or in admixture with excipients, or in the form ofelixirs, solutions or suspensions containing flavouring or colouringagents. They can also be injected parenterally, for exampleintravenously, intramuscularly or subcutaneously. For parenteraladministration, they are best used in the form of a sterile aqueoussolution which may contain other substances, for example enough salts orglucose to make the solution isotonic with blood. For buccal orsublingual administration they may be administered in the form oftablets or lozenges which can be formulated in a conventional manner.

For oral, parenteral, buccal and sublingual administration to patients,the daily dosage level of the compounds of formula (I) and theirpharmaceutically acceptable salts and solvates will be from 1 to 1000 mg(in single or divided doses). Thus tablets or capsules may contain from0.5 to 500 mg of active compound for administration singly, or two ormore at a time, as appropriate. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual patient and it will vary with the age, weight and response ofthe particular patient. The above dosages are exemplary of the averagecase; there can, of course, be individual instances where higher orlower dosage ranges are merited and such are within the scope of thisinvention.

Thus the invention provides a pharmaceutical composition comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,or a pharmaceutically acceptable solvate of either entity, together witha pharmaceutically acceptable diluent or carrier.

The invention also provides a compound of formula (I), or apharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable solvate of either entity, or a pharmaceutical compositioncontaining any of the foregoing, for use as a medicament.

The invention further includes the use of a compound of formula (I), ora pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable solvate of either entity, or a pharmaceutical compositioncontaining any of the foregoing, for the manufacture of a medicament forthe curative or prophylactic treatment of deep vein thrombosis (DVT)after surgery, major medical illness, paralysis, malignancy, prolongedimmobilisation trauma, application of lower limb plaster casts, orfractures of the lower limbs or pelvis; recurrent DVT; DVT duringpregnancy when there is a previous history thereof; reocclusionfollowing thrombolytic therapy; chronic arterial obstruction; peripheralvascular disease; acute myocardial infarction; unstable angina; atrialfibrillation; thrombotic stroke; transient ischaemic attacks;disseminated intravascular coagulation; coagulation in extracorporealcircuits; occlusion of arterio-venous shunts and blood vessel grafts(including coronary artery by-pass grafts); restenosis and occlusionfollowing angioplasty; neurodegenerative disorders; inflammatorydisorders; or scarring.

In a further aspect, the invention provides a method of treating amammal (including a human being) to cure or prevent deep vein thrombosis(DVT) after surgery, major medical illness, paralysis, malignancy,prolonged immobilisation trauma, application of lower limb plastercasts, or fractures of the lower limbs or pelvis; recurrent DVT; DVTduring pregnancy when there is a previous history thereof; reocclusionfollowing thrombolytic therapy; chronic arterial obstruction; peripheralvascular disease; acute myocardial infarction; unstable angina; atrialfibrillation; thrombotic stroke; transient ischaemic attacks;disseminated intravascular coagulation; coagulation in extra-corporealcircuits; occlusion of arterio-venous shunts and blood vessel grafts(including coronary artery by-pass grafts); restenosis and occlusionfollowing angioplasty; neurodegenerative disorders; inflammatorydisorders; or scarring; which comprises treating said mammal with aneffective amount of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, or a pharmaceutically acceptable solvate ofeither entity, or a pharmaceutical composition containing any of theforegoing.

The syntheses of the compounds of the invention and of the intermediatesf or us e therein are illustrated by the following Examples andPreparations. The purity (Rf) of the compounds was routinely monitoredby thin layer chromatography using Merck Kieselgel 60 F₂₅₄ plates andthe following solvent systems (SS):

1. isobutyl methyl ketone:glacial acetic acid:water, 2:1:1 (upperphase);

2. hexane:ethyl acetate, 1:1;

3. hexane:ethyl acetate, 7:3;

4. dichloromethane:methanol:0.880 aqueous ammonia, 85:15:2;

5. dichloromethane:methanol:0.880 aqueous ammonia, 84:14:2;

6. hexane:ethyl acetate, 6:4;

7. dichloromethane:methanol:0.880 aqueous ammonia, 93:7:1;

8. dichloromethane:methanol, 90:10;

9. dichloromethane:methanol:0.880 aqueous ammonia, 93:7:2;

10. dichloromethane:methanol:0.880 aqueous ammonia, 90:10:1;

11. dichloromethane:methanol, 95:5;

12. dichloromethane:methanol:0.880 aqueous ammonia, 193:7:1;

13. ethyl acetate;

14. hexane:ether, 1:1;

15. hexane:ether, 1:3;

16. dichloromethane:methanol:0.880 aqueous ammonia, 80:20:5;

17. chloroform:methanol, 95:5;

18. hexane:ethyl acetate, 3:7;

19. methanol:ethyl acetate:glacial acetic acid:0.880 aqueousammonia:water, 60:12:4:4:8.

¹ H Nuclear magnetic resonance (NMR) spectra were recorded using eithera Nicolet QE-300 or a Bruker AC-300 spectrometer and were in all casesconsistent with the proposed structures.

Mass spectra were obtained with a Fisons Instrument Trio 1000spectrometer using thermospray ionisation.

Room temperature means 20-25° C.

EXAMPLE 1 N- N-(3-Methyl-3-cyclohexenyl)-(S)-α-asrpartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

A stirred, ice-cooled solution of the title compound of Preparation 20(111 mg, 0.54 mmol) in dichloromethane (10 ml) was saturated withhydrogen chloride, the cooling bath removed and the resulting solutionstirred at room temperature until total deprotection was complete:(typically 2 to 6 hours at room temperature). Evaporation under reducedpressure gave the title compound (76 mg) as a white foam. Rf 0.60 (SS19). Found: C,46.98; H,8.11; N, 11.13. C₂₄ H₄₁ N₅ O₄ ; 2HCl; 3H₂ O; 0.40CH₂ Cl₂ requires C,46.92; H,8.04; N,11.21%

EXAMPLE 2 N- N-(N-Methyl-3-piperidyl)-(S) α-aspartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine trihydrochloride

Obtained from the title compound of Preparation 22 by analogy withExample 1. Rf 0.18 (SS 19). Found: C,41.13; H,7.29; N,12.26. C₂₃ H₄₂ N₆O₄ ; 3HCl; 1.50 H₂ O; 1.20 CH₂ Cl₂ requires C,41.23; H,7.21; N,11.92%.m/e 467.5 (M+H)⁺.

EXAMPLE 3 N- N-(2-Norbornyl)-(S)α-aspartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 23 by analogy withExample 1. Rf 0.52 (SS 19). Found: C,49.78; H,8.02; N,11.97. C₂₄ H₄₁ N₅O₄ ; 2HCl; H₂ O; 0.40 CH₂ Cl₂ requires C,49.80; H,7.84; N,11.90%. m/e464.3 (M+H)⁺.

EXAMPLE 4 N-(N-Cyclopentylmethyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)-ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 39 by analogy withExample 1. Rf 0.56 (SS 19). Found: C,48.61; H,7.94; N,12.07. C₂₃ H₄₁ N₅O₄ ; 2HCl; 1.50 H₂ O; 0.33 CH₂ Cl₂ requires C,48.35; H,8.11; N,12.08%.m/e 452 (M+H)⁺.

EXAMPLE 5 N- N-(cis-3,5-Dimethylcyclohexyl)-(S)-α-aspartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 30 (diastereoisomer A)by analogy with Example 1. Rf 0.59 (SS 19). Found: C,50.10; H,8.24;N,11.24. C₂₅ H₄₅ N₅ O₄ ; 2HCl; 2.30 H₂ O; 0.10 CH₂ Cl₂ requires C,50.04;H,8.67; N,11.62%. m/e 480 (M+H)⁺.

EXAMPLE 6 N- N-(cis-3,5-Dimethylcyclohexyl)-(S)-α-aspartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 30 (diastereoisomer B)by analogy with Example 1. Rf 0.59 (SS 19). Found: C,49.69; H,8.54;N,11.19. C₂₅ H₄₅ N₅ O₄ ; 2HCl; 2.50 H₂ O; 0.10 CH₂ Cl₂ requires C,49.74;H,8.68; N,11.55%. m/e 480 (M+H)⁺.

EXAMPLE 7 N- N-(3,3-Dimethylcyclohexyl)-(S)-α-aspartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 27 by analogy withExample 1. Rf 0.59 (SS 19). Found: C,50.27; H,8.75; N,11.53. C₂₅ H₄₅ N₅O₄ ; 2HCl; 2H₂ O; 0.15 CH₂ Cl₂ requires C,50.23; H,8.60; N,11.65%. m/e480 (M+H)⁺.

EXAMPLE 8 N-(N-Cycloheptyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 31 by analogy withExample 1. Rf 0.50 (SS 19). Found: C,48.45; H,8.21; N,11.21. C₂₄ H₄₃ N₅O₄ ; 2HCl; 1.10 H₂ O; 0.60 CH₂ Cl₂ requires C,48.49; H,8.01; N,1 1.49%.m/e 466.5 (M+H)⁺.

EXAMPLE 9 N- N-(N-Methyl-4-piperidyl)-(S)-α-aspartyl!-2(R,S)-2-(N-amidino4-piperidyloxy)ethyl!piperidine trihydrochloride

Obtained from the title compound of Preparation 19 by analogy withExample 1. Rf 0.13 (SS 19). Found: C,43.79; H,7.59; H,12.43. C₂₃ H₄₂ N₆O₄ ; 3HCl; 1.40 H₂ O; 0.60 CH₂ Cl₂ requires C,43.46; H,7.57; N,12.89%.m/e 467.5 (M+H)⁺.

EXAMPLE 10 N-(N-Cyclohexylmethyl-(S)-α-aspartyl)-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 38 by analogy withExample 1. Rf 0.56 (SS 19). Found: C,48.80; H,8.29; N,11.48. C₂₄ H43N₅O₄ ; 2HCl; 1.50 H₂ O; 0.40 CH₂ Cl₂ requires C,48.88; H,8.20; N, 11.68%.m/e 466 (M+H)⁺.

EXAMPLE 11 N-(N-Cyclohexyl-(S)-α-aspartyl)-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 25 by analogy withExample 1. Rf 0.57 (33 19). Found: C,47.51; H,7.75; N,11.62. C₂₃ H₄₁ N₅O₄ ; 2HCl; CH₂ Cl₂ requires C,47.28; H,7.44; N,11.49%.

EXAMPLE 12 N- N-(4-Tetrahydropyranyl)-(S)-α-aspartyl!-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 26 by analogy withExample 1. Rf 0.48 (SS 19). Found: C,44.44; H,7.27; N,11.15. C₂₂ H₃₉ N₅O₅ ; 2HCl; 0.50 H₂ O; CH₂ Cl₂ requires C,44.53; H,7.15; N,11.29%. m/e454 (M+H)⁺.

EXAMPLE 13 N- N-(3.3-Dimethylcyclohexyl)-(S)-α-aspartyl!-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 28 by analogy withExample 1. Rf 0.60 (SS 19). Found: C,49.10; H,8.24; N,11.17. C₂₅ H₄₅ N₅O₄ ; 2HCl; H₂ O; 0.67 CH₂ Cl₂ requires C,49.18; H,8.09; N,11.18%. m/e480 (M+H)⁺.

EXAMPLE 14 N-(N-Cyclopentyl-(S)-α-aspartyl)-2(R,S)-2-(N-amidino-4-piperidyloxy)-ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 33 by analogy withExample 1. Rf 0.44 (SS 19). Found: C,46.53; H,7.88; N,11.82. C₂₂ H₃₉ N₅O₄ ; 2HCl; 1.50 H₂ O; 0.50 CH₂ Cl₂ requires C,46.60; H,7.82; N,12.97%.m/e 438 (M+H)⁺.

EXAMPLE 15 N-(N-Cycloheptyl-(S)-α-aspartyl!-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 32 by analogy withExample 1. Rf 0.49 (SS 19). Found: C,47.42; H,8.07; N,11.29. C₂₄ H₄₃ N₅O₄ ; 2HCl; 1.25 H₂ O; 0.75 CH₂ Cl₂ requires C,47.58; H,7.91; N,1 1.21%.m/e 466 (M+H)⁺.

EXAMPLE 16 N-(N-Cyclooctyl-(S)-α-aspartyl)-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 34 by analogy withExample 1. Rf 0.58 (SS 19). Found: C,49.60; H,8.56; N,11.19. C₂₅ H₄₅ N₅O₄ ; 2HCl; 2H₂ O; 0.25 CH₂ Cl₂ requires C,49.73; H,8.51; N,11.48%.

EXAMPLE 17 N- N-(2-Hydroxycyclohexyl)-(S)-α-aspartyl!-2(R,S)-2-(N-amidino-4-Piperidyloxy)ethyl!piperidine bis-trifluoroacetate

Obtained from the title compound of Preparation 35 by treatment with a1:1 mixture of trifluoroacetic acid and dichloromethane (200 μl) for 3hours at room temperature, followed by evaporation of the reactionmixture, to provide 0.37 mg. m/e 426.4 (M-C(NH)NH₂ +H)⁺.

EXAMPLE 18 N- N-(3-Tetrahydrothiopyranyl)-(S)-α-aspartyl!-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine bis-trifluoroacetate

Obtained from the title compound of Preparation 36 by analogy withExample 17 to provide 0.39 mg. m/e 428.3 (M-C(NH)NH₂ +H)⁺.

EXAMPLE 19 N- N-(4-Tetrahydrothiopyranyl)-(S)a-aspartyl!-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine bis-trifluoroacetate

Obtained from the title compound of Preparation 37 by analogy withExample 17 to provide 2 mg.

EXAMPLE 20 N-(N-Cyclohexyl-(S)-α-glutamyl)-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 52 by analogy withExample 1. Rf 0.49 (SS 19). Found: C,48.53; H,7.63; N,11.40. C₂₄ H₄₃ N₅O₄ ; 2HCl; H₂ O; 0.65 CH₂ Cl₂ requires C,48.40; H,7.96; N,11.45%. m/e466 (M+H)⁺.

EXAMPLE 21 N-N-Methyl-N-(cis-3,5-dimethylcyclohexyl)-(S)-α-aspartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihyhdrochloride

Obtained from the title compound of Preparation 45 by analogy withExample 1. Rf 0.61 (SS 19). Found: C,50.39; H,8.57; N,11.39. C₂₆ H₄₇ N₅O₄ ; 2HCl; 1.50 H₂ O; 0.40 CH₂ Cl₂ requires C,50.52; H,8.48; N, 1.16%.m/e 494 (M+H)⁺.

EXAMPLE 22 N-(N-Cyclohexyl-N-methyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 40 by analogy withExample 1. Rf 0.50 (SS 19). Found: C,47.31; H,8.02; N,10.74. C₂₄ H₄₃ N₅O₄ ; 2HCl; 1.30 H₂ O; 0.80 CH₂ Cl₂ requires C,47.27; H,7.90; N,11.11%.m/e 466 (M+H)⁺.

EXAMPLE 23 N- N-Methyl-N-(3-methyl-3-cyclohexenyl-(S)-α-aspartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 46 by analogy withExample 1. Rf 0.50 (SS 19). Found: C,48.01; H,7.82; N,11.00. C₂₅ H₄₃ N₅O₄ ; 2HCl; 1.10 H₂ O; 0.90 CH₂ Cl₂ requires C,48.09; H,7.64; N,10.83%.m/e 478.9 (M+H)⁺.

EXAMPLE 24 N-(N-Cycloheptyl-N-methyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 43 by analogy withExample 1. Rf 0.52 (SS 19). Found: C,49.77; H,8.12; N,11.47%. C₂ H₄₅ N₅O₄ ; 2HCl; 2H₂ O; 0.25 CH₂ Cl₂ requires C,49.73; H,8.51; N,11.48%. m/e480.2 (M+H)⁺.

EXAMPLE 25 N- N-Methyl-N-(3,3-dimethylcyclohexyl)-(S)-α-aspartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 42 by analogy withExample 1. Rf 0.60 (SS 19). Found: C,50.18; H,8.49; N,10.84. C₂₅ H₄₇ N₅O₄ ; 2HCl; 2.50 H₂ O; 0.20 CH₂ Cl₂ requires C,50.06; H,8.72; N,11.14%.m/e 494 (M+H)⁺.

EXAMPLE 26 N-(N-Cyclohexyl-N-methyl-(S)-α-aspartyl)-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 41 by analogy withExample 1. Rf 0.58 (SS 19). Found C,47.80; H,7.89; N,11.03. C₂₄ H₄₃ N₅O₄ ; 2HCl; 0.50 H₂ O; CH₂ Cl₂ requires C,47.47; H,7.65; N,11.07%. m/e466 (M+H)⁺.

EXAMPLE 27 N-(N-Cyclopentyl-N-methyl-(S)-α-aspartyl)-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 47 by analogy withExample 1. Rf 0.46 (SS 19). Found: C,48.06; H,7.76; N,1 1.67. C₂₃ H₄₁ N₅O₄ ; 2HCl; H₂ O; 0.50 CH₂ Cl₂ requires C,48.25; H,7.93; N,11.97%. m/e452 (M+H)⁺.

EXAMPLE 28 N-(N-Cycloheptyl-N-methyl-(S)-α-aspartyl)-2(R,S)-2-(N-amidino-4-piperidyloxy)-ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 44 by analogy withExample 1. Rf 0.52 (SS 19). Found: C,46.93; H,7.88; N,10.64. C₂₅ H₄₅ N₅O₄ ; 2HCl; H₂ O; CH₂ Cl₂ requires C,47.21; H,7.77; N,10.55%. m/e 480(M+H)⁺.

EXAMPLE 29 N-(N-Cyclohexyl-N-ethyl-(S)-α-aspartyl)-2(R,S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 48 by analogy withExample 1. Rf 0.54 (SS 19). Found: C,48.54; H,7.88; N,10.61. C₂₅ H₄₅ N₅O₄ ; 2HCl; 0.10 H₂ O; CH₂ Cl₂ requires C,48.85; H,7.76; N,10.95%. m/e480.7 (M+H)⁺.

EXAMPLE 30 N- (2(S)-Carboxymethyl)piperidinoacetyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 49 by analogy withExample 1. Rf 0.45 (SS 19). Found: C,44.68; H,7.62; N,1 1.32. C₂₂ H₃₉ N₅O₄ ; 2HCl; 1.50 H₂ O; 0.90 CH₂ Cl₂ requires C,44.80; H,7.52; N,11.41%.

EXAMPLE 31 N-(N-Cyclohexyl-(S)-α-aspartyl)-2(R,S)-2-(4-amidinophenoxy)ethyl!piperidine dihydrochloride

A 1M aqueous solution of sodium hydroxide (4.2 ml, 4.2 mmol) was addedto a stirred suspension of the title compound of Preparation 59 (390 mg,0.71 mmol) in 1,4-dioxan (4.2 ml). After a further 50 minutes, theresulting solution was acidified to pH3 with 1M hydrochloric acid andevaporated under reduced pressure; residual solvents were removed byazeotropy using 2-propanol. The resulting residue was extracted with hot2-propanol, then the combined extracts filtered and evaporated underreduced pressure to afford the title compound (290 mg) as a white solid.Rf (diastereoisomers) 0.08 and 0.12 (SS 16). Found: C,54.81; H,7.97;N,9.61. C₂₄ H₃₆ N₄ O₄ ; 2HCl; 0.70 H₂ O; CH₃ CH(OH)CH₃ requires C,54.95;H,8.09; N,9.49%. m/e 445 (M+H)⁺.

EXAMPLE 32 N- N-(N-Methyl-4-piperidyl)-(S)-α-aspartyl!-2(R,S)-2-(4-amidinophenoxy)ethyl!-piperidine trihydrochloride

Obtained from the title compound of Preparation 61 by analogy withExample 31. Rf 0.13 (SS 19). Found: C,48.57; H,7.52; N,10.74. C₂₄ H₃₇ N₅O₄ ; 3HCl; 1.90 H₂ O; 0.40 CH₃ CH(OH)CH₃ requires C,48.26; H,7.55;N,11.16%. m/e 443 (M+H-NH₃)+and 460 (M+H)⁺.

EXAMPLE 33 N- N-Methyl-N-(N-methyl-4-piperidyl)-(S)-α-aspartyl!-2(R,S)-2-(4-amidinophenoxy)ethyl!piperidine trihydrochloride

Obtained from the title compound of Preparation 66 by analogy withExample 31. Rf 0.13 (SS 19). Found: C,50.10; H,8.40; N,9.78. C₂₅ H₃₉ N₅O₄ ; 3 HCl; 2H₂ O; 1.20 CH₃ CH(OH)CH₃ ; 0.30 (C₂ Hs)₂ 0 requiresC,50.17; H,8.27; N,9.81%. m/e 457 (M+H-NH₃)+and 474 (M+H)⁺.

EXAMPLE 34 N-(N-4-Cycloheptenyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)-ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 67 by analogy withExample 1. Rf 0.60 (SS 19). Found: C,49.98; H,7.92; N,11.84. C₂₄ H₄₁ N₅O₄ ; 2HCl; 1.20 H₂ O; 0.30 CH₂ Cl₂ requires C,50.01; H,7.94; N,12.00%.

EXAMPLE 35 N-(N-4-Cycloheptenyl-N-methyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dichhydrochloride

Obtained from the title compound of Preparation 68 by analogy withExample 1. Rf 0.54 (SS 19). Found: C,48.45; H,7.69; N,10.86. C₂₅ H₄₃ N₅O₄ ; 2HCl; 0.50 H₂ O; CH₂ Cl₂ requires C,48.45; H,7.51; N,10.87%. m/e478 (M+H)⁺.

EXAMPLE 36 N-(N-Cyclopropylmethyl-(S)-αaspartyl)-2(S)-2-(N-amidine-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 69 by analogy withExample 1. Rf 0.58 (SS 19). Found: C,45.14; H,7.84; N,12.19. C₂₁ H₃₇ N₅O₄ ; 2HCl; 2H₂ O; 0.40 CH₂ Cl₂ requires C,45.37; H,7.79; N,12.36%. m/e424 (M+H)⁺.

EXAMPLE 37 N-(N-3-Pentyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 70 by analogy withExample 1. Found: C,44.29; H,8.49; N,11.13. C₂₂ H₄₁ N₅ O₄ ; 2HCl; 3H₂ O;0.50 CH₂ Cl₂ requires C,44.37; H,8.28; N,11.50%. m/e 440.6 (M+H)⁺.

EXAMPLE 38 N-(N-2-Cyclohexyl-N-methyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-(piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 72 by analogy withExample 1. Rf 0.57 (SS 19). Found: C,48.91; H,8.15; N,11.90. C₂₄ H₄₁ N₅O₄ ; 2HCl; 3H₂ O requires C,48.81; H,8.36; N,1 1.86%. m/e 464.3(M+H)⁺.

EXAMPLE 39 N-(N-3(R)-Methylcyclohexyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the mixture of diastereoisomers of the title compound ofPreparation 73 by analogy with Example 1. Rf 0.55 (SS 19). Found:C,48.97; H,8.18; N,11.54. C₂₄ H₄₃ N₅ O₄ ; 2HCl; 1.50 H₂ O requiresC,48.88; H,8.20; N,11.68%. m/e 466.5(M+H)⁺.

EXAMPLE 40

N-(N-Methyl-N-3(R)-methylcyclohexyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the mixture of diastereoisomers of the title compound ofPreparation 74 by analogy with Example 1. Rf 0.51 (SS 19). Found:C,50.26; H,8.59; N,1 1.69. C₂₅ H₄₅ N₅ O₄ ; 2HCl; 2.60 H₂ O requiresC,50.09; H,8.78; N, 11.68%. m/e 480.3 (M+H)⁺.

A single diastereoisomer of the title compound was also obtained byeffecting the corresponding deprotection of the single diastereoisomerfrom Preparation 74. Rf 0.57 (SS 19). m/e 480.2 (M+H)⁺.

EXAMPLE 41 N- N-Methyl-N-(2-norbornyl-(S)-α-aspartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 75 by analogy withExample 1. Rf 0.64 (SS 19). Found: C,50.39; H,8.09; N,11.52. C₂₅ H₄₁ N₅O₄ ; 2HCl; 2H₂ O; 0.22 CH₂ Cl₂ requires C,50.21; H,7.93; N,11.61%. m/e492.2 (M+NH₄)⁺.

EXAMPLE 42 N- N-(3-Tetrahydropyranyl)-(S)-α-aspartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 77 by analogy withExample 1. Rf 0.53 (SS 19). Found: C,44.51; H,7.28; N,11.23. C₂₂ H₃₉ N₅O₅ ; 2HCl; 2H₂ O; 0.50 CH₂ Cl₂ requires C,44.67; H,7.66; N,11.58%. m/e454.2 (M+H)⁺.

EXAMPLE 43 N- N-Methyl-N-(3-tetrahydropyranyl)-(S)-α-aspartyl!-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 78 by analogy withExample 1. Rf 0.52 (SS 19). Found: C,43.46; H,8.24; N,10.92. C₂₃ H₄₁ N₅O₅ ; 2HCl; 3.10 H₂ O; 0.60 CH₂ Cl₂ requires C,43.79; H,8.02; N,10.82%.

EXAMPLE 44 N-(N-3-Cyclohexenyl-(S)-a-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 80 by analogy withExample 1. Rf 0.47 (SS 19). Found: C,47.32; H,7.76; N,11.28. C₂₃ H₃₉ N₅O₄ ; 2HCl; 2H₂ O; 0.50 CH₂ Cl₂ requires C,46.96; H,7.71; N,11.65%. m/e450.0 (M+H)⁺.

EXAMPLE 45 N-(N-3-Cyclohexenyl-N-methyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine dihydrochloride

Obtained from the title compound of Preparation 81 by analogy withExample 1. Rf 0.75 (SS 19). Found: C,46.46; H,8.20; N,10.85. C₂₄ H₄₁ N₅O₄ ; 2HCl; 3H₂ O; 0.50 CH₂ Cl₂ requires C,46.48; H,7.96; N,1 1.06%. m/e464.3 (M+H)⁺.

PREPARATION 1 2(S)-(2-Hydroxyethyl)piperidine

2(R,S)-(2-Hydroxyethyl)piperidine was resolved using(1S)-(+)-10-camphorsulphonic acid as described in Rec. Trav. chim.,1971, 90, 755 via the intermediate (S),(S)-10-camphorsulphonate salt,m.p. 167° C. (lit. 166-167° C.), α!_(D) ²⁵ +32.5° (c=2.2, CHCl₃)(reported in J. Amer. Chem. Soc., 1960, 82, 2613 as α!_(D) 32.40 (c=2,CHCl₃)). The absolute configuration of the salt was determined by X-raycrystallographic analysis.

The title compound was obtained as fine needles, m.p. 69-70° C. (lit.68-69° C.), Rf 0.25 (SS 1). GC analysis of the bis-trifluoroacetylderivative, using a Chiraldex B-TA No C70 column, showed an enantiomericexcess (ee) of >98%.

PREPARATION 2 N-Benzyloxycarbonyl-2(S)-(2-hydroxyethyl)piperidine

To a stirred, ice-cooled solution of the title compound of Preparation 1(13.4 g, 0.104 mol) in dichloromethane (250 ml) were added,sequentially, triethylamine (15.9 ml, 0.114 mol) andN-(benzyloxycarbonyloxy)succinimide (27.21 9, 0.109 mol). The coolingbath was removed, then the reaction mixture stirred at room temperaturefor 18 hours, washed with brine, dried (Na₂ SO₄) and evaporated underreduced pressure to give an oil (30.6 g) which was purified bychromatography on silica gel, using hexane:ethyl acetate (1:1) aseluant, to provide the title compound (27.5 g) as an oil. Rf 0.48 (SS2). α!_(D) ²⁵ -24.5° (c=1.06, CH₃ OH).

PREPARATION 3 N-Benzyloxycarbonyl-2(R,S)-(2-hydroxyethyl)piperidine

Obtained from 2(R,S)-(2-hydroxyethyl)piperidine by analogy withPreparation 2. Rf 0.48 (SS 2). Found: C,68.27; H,8.26; N,5.28. C₁₅ H₂₁NO₃ requires C,68.42; H,8.04; N,5.32%. m/e 264.4 (M+H)⁺.

PREPARATION 4N-Benzyloxycarbonyl-2(S)-(2-methanesulphonyloxyethyl)piperidine

Methanesulphonyl chloride (16.1 ml, 0.208 mol) was added dropwise over15 minutes to a stirred, ice-cooled solution of the title compound ofPreparation 2 (27.43 g, 0.104 mol) and triethylamine (29 ml, 0.208 mol)in dichloromethane (350 ml), the temperature of the reaction mixturebeing allowed to rise to 17° C. during the addition. After a further 25minutes, the reaction mixture was washed with 1M aqueous citric acidsolution, water and saturated aqueous sodium bicarbonate solution, dried(Na₂ SO₄) and evaporated under reduced pressure to give a light yellowoil (40 g) which was purified by chromatography on silica gel, usinghexane:ethyl acetate (1:1) as eluant, to furnish the title compound(33.5 g) as a clear oil which solidified on standing. Rf 0.59 (SS 8).

A trace of ethyl acetate was removed from the product azeotropically,using hexane, before the next step of the reaction sequence.

PREPARATION 5N-Benzyloxycarbonyl-2(R,S)-(2-methanesulphonyloxyethyl)piperidine

Obtained from the title compound of Preparation 3 by analogy withPreparation 4. Rf 0.50 (SS 8). Found: C,54.76; H,6.35; N,4.10. C₁₆ H₂₃NO₅ S; 0.15 CH₂ Cl₂ requires C,54.77; H,6.63; N,3.95%.

PREPARATION 6 N-t-Butoxycarbonyl-4-hydroxypiperidine

Di-t-butyl dicarbonate (35.58 g, 0.163 mol) was added to a stirred,ice-cooled solution of 4-hydroxypiperidine (15.0 g, 0.148 mol) indichloromethane (250 ml). The cooling bath was removed, then thereaction mixture stirred at room temperature for 56 hours, washed with 1M aqueous citric acid solution, dried (MgSO₄) and evaporated underreduced pressure to give a yellowish oil, treatment of which with hexane(20 ml), followed by chilling, promoted crystallisation. Filtration andwashing of the product with cold hexane afforded the title compound(25.72 g). Rf 0.37 (SS 2). Found: C,59.27; H,9.70; N,6.96. C₁₀ H₁₉ NO₃requires C, 59.68; H,9.51; N,6.96%.

PREPARATION 7 N-Benzyloxycarbonyl-2(S)- 2-(N-t-butoxycarbonyl-4-piperidyloxy)ethyl!piperidine

The title compound of Preparation 6 (19.67 g, 97.5 mmol) was added to astirred suspension of sodium hydride (60% dispersion in oil, 3.9 g, 97.5mmol) in dry dimethylformamide (150 ml) under nitrogen. After 2 hours, asolution of the title compound of Preparation 4 (32.9 g, 96.4 mmol) indry dimethylformamide (100 ml) was added and the resulting reactionmixture stirred for 18 hours. The bulk of the solvent was removed underreduced pressure, the residue diluted with water and the resulting oilysuspension extracted with ethyl acetate (×3). The combined extracts werewashed with brine, dried (Na₂ SO₄) and evaporated under reduced pressureto give an oil which was purified by chromatography on silica gel, usinghexane:ethyl acetate (7:3) as eluant, to afford the title compound(26.26 g) as an oil. Rf 0.35 (SS 3). α!_(D) ²⁵ -12.3° (c=1.01, CH₃ OH).Found: C,67.07; H,8.75; N,6.04. C₂₅ H₃₈ N₂ O₅ requires C,67.24; H,8.58;N,6.27%.

PREPARATION 8 N-Benzyloxycarbonyl-2(R,S)-2-(N-t-butoxycarbonyl-4-piperidyloxy)ethyl!piperidine

Obtained from the title compound of Preparation 5 by analogy withPreparation 7. Rf 0.30 (SS 3).

PREPARATION 9 N-Benzyloxycarbonyl-2(S)-2-(4-piperidyloxy)ethyl!piperidine hydrochloride

A stirred, ice-cooled solution of the title compound of Preparation 7(26.16 g, 58.6 mmol) in dichloromethane (300 ml) was saturated withhydrogen chloride. After a further 1.25 hours the solvent was removed byevaporation under reduced pressure and the residual hydrogen chlorideremoved azeotropically using dichioromethane to give the title compound(22.59 g) as a white foam. Rf 0.60 (SS 4). m/e 347.0 (M+H)⁺.

PREPARATION 10 N-Benzyloxycarbonyl-2(R,S)-2-(4-piperidyloxy)ethyl!piperidine hydrochloride

Obtained from the title compound of Preparation 8 by analogy withPreparation 9. Rf 0.50 (SS 5). Found: C,62.06; H,7.71; N,6.94. C₂₀ H₃₀N₂ O₃ ; HCl requires C,62.27; H,7.89; N,7.32%. m/e 347.0 (M+H)⁺.

PREPARATION 11 N-Benzyloxycarbonyl-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Triethylamine (24.5 ml, 0.176 mol) was added to a stirred, ice-cooledsolution of the title compound of Preparation 9 (22.59 g, 58.6 mmol) indichloromethane (280 ml), the mixture allowed to warm to roomtemperature and then N,N'-di-t-butoxycarbonyl-S-methylisothiourea (J.Med. Chem., 1993, 36, 2956; 18.7 g, 64.4 mmol) and mercuric chloride(15.91 g, 58.6 mmol) added sequentially. The reaction mixture wasstirred for 18 hours, for a further 2 hours under reflux, then filteredusing an appropriate filter aid. The filtrate was washed with water,during which further filtration to remove precipitated material wasnecessary, dried (Na₂ SO₄) and evaporated under reduced pressure to givecrude product which was purified by chromatography on silica gel, usingan elution gradient of hexane:ethyl acetate (7:3 to 1:1), to provide thetitle compound (30.37 g) as a gummy foam. Rf 0.20 (SS 2). α!_(D) ²⁵-5.7° (c=1.1, CH₃ OH). Found: C,62.42; H,8.30; N,9.31. C₃₁ H₄₈ N₄ O₇requires C,62.22; H,8.13; N,9.18%.

PREPARATION 12 N-Benzyloxycarbonyl-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 10 by analogy withPreparation 11. Rf 0.35 (SS 6). m/e 589.4 (M+H)⁺.

PREPARATION 13 2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

A solution of the title compound of Preparation 11 (26.82 g, 45.55 mmol)in absolute ethanol (400 ml) was hydrogenated over 10% palladium oncharcoal (5.0 g) at room temperature and 414 kPa (60 psi) for 2.5 hours.The resulting mixture was filtered using a filter aid and the filtrateevaporated under reduced pressure to furnish the title compound (20.36g) as a gum. Rf 0.13 (SS 7). α!_(D) ²⁵ -1.69° (c=1.3, CH₃ OH). m/e 455(M+H)⁺.

PREPARATION 14 2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 12 by analogy withPreparation 13. Rf 0.22 (SS 7).

PREPARATION 15 N-N-(9-Fluorenylmethoxycarbonyl)-O-t-butyl-(S)-α-aspartyl-2(S)-{2N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

N-Ethyldiisopropylamine (1.27 ml, 4.6 mmol) was added to a stirred,ice-cooled solution of the title compound of Preparation 13 (1.05 g, 2.3mmol), N-fluoroenylmethoxycarbonyl-(S)-aspartic acid β-t-butyl ester(946 mg, 2.3 mmol) and bromotris(pyrrolidino)phosphoniumhexafluorophosphate (1.18 g, 2.5 mmol) in dry dichloromethane (6 ml).After 2.5 hours more coupling reagent (118 mg, 0.25 mmol) was added andstirring continued for a further 0.5 hour. The reaction mixture wasdiluted with ethyl acetate, washed sequentially with water, 1M aqueouscitric acid, water, saturated aqueous sodium bicarbonate solution andbrine, dried (Na₂ SO₄) and evaporated under reduced pressure to givecrude product (2.87 g) which was purified by chromatography on silicagel, using hexane:ethyl acetate (1:1) as eluant, to furnish the titlecompound (1.58 g) as a white foam. Rf 0.30 (SS 2). Found: C,64.42;H,8.05; N,8.24, C₄₆ H₆₅ N₅ O₁₀ ; 0.40 CH₃ CO₂ CH₂ CH₃ requires C,64.73;H,7.78; N,7.93%. m/e 626 (M-Fmoc+H)⁺.

PREPARATION 16 N-N-(9-Fluorenylmethoxycarbonyl)-O-t-butyl-(S)-α-aspartyl!-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 14 by analogy withPreparation 15. Rf 0.68 (SS 8). m/e 849 (M+H)⁺.

PREPARATION 17 N-(O-t-Butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Piperidine (1.7 ml, 18.3 mmol) was added to a stirred solution of thetitle compound of Preparation 15 (1.55 g, 1.83 mmol) in tetrahydrofuran(7.5 ml). After 1 hour, the reaction mixture was evaporated underreduced pressure and the residue purified by chromatography on silicagel, using dichloromethane: methanol:0.880 aqueous ammonia (193:7:1) aseluant, to afford the title compound (1.05 g) as a white foam. Rf 0.50(SS 7). Found: C,58.93; H,9.08; N,11.12. C₃₁ H₅₅ N₅ O₈ ; 0.30 H₂ Orequires C,58.99; H,8.88; N,11.09%. m/e 626 (M+H)⁺.

PREPARATION 18 N-(O-t-Butyl-(S)-α-aspartyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 16 by analogy withPreparation 17. Rf 0.52 (SS 8). m/e 626 (M+H)⁺.

PREPARATION 19 N-N-(N-Methyl-4-piperidyl)-O-t-butyl-(S)-α-aspartyl!-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

To a stirred solution of the title compound of Preparation 18 (500 mg,0.80 mmol) in anhydrous tetrahydrofuran (5 ml) was addedN-methyl-4-piperidone (0.11 ml, 0.90 mmol) followed by glacial aceticacid (96 μl, 1.66 mmol). After a further 5 minutes sodiumtriacetoxyborohydride (254 mg, 1.12 mmol) was added and the reactionmixture stirred for 18 hours, then diluted with ethyl acetate (120 ml),washed with saturated aqueous sodium bicarbonate solution and brine,dried (MgSO₄) and evaporated under reduced pressure to give the crudeproduct which was purified by chromatography on silica gel, using anelution gradient of dichloromethane:methanol:0.880 aqueous ammonia(100:0:0 to 100:5:0 to 200:15:1 to 200:15:2), to provide the titlecompound (0.51 g) as a white foam. Rf 0.50 (SS 9). Found: C,60.59;H,9.15; N,11.58. C₃₇ H₆₆ N₆ O₈ ; 0.20 CH₂ Cl₂ requires C,60.38; H,9.04;N,11.36%.

PREPARATION 20 N-N-(3-Methyl-3-cyclohexenyl)-O-t-butyl-(S)-α-aspartyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 17 and3-methyl-3-cyclohexenone (J. Org. Chem., 1977, 42, 1051) by analogy withPreparation 19. Rf 0.96 (SS 10). Found: C,61.67; H,9.07; N,9.21. C₃₈ H₆₅N₅ O₈ ; 0.25 CH₂ Cl₂ requires C,61.98; H,8.91; N,9.45%. m/e 720.3(M+H)⁺.

PREPARATION 21 N-Methyl-3-piperidone

A solution of anhydrous dimethyl sulphoxide (16.3 ml, 236 mmol) indichloromethane (43 ml) was added, dropwise, to a stirred solution ofoxalyl chloride (9.45 ml, 109 mmol) in dichloromethane at about -60° C.under nitrogen followed, 2 minutes later, by a solution ofN-methyl-3-hydroxypiperidine (11.52 g, 100 mmol) in dichloromethane (100ml). After a further 10 minutes, triethylamine (68.9 ml, 497 mmol) wasalso added dropwise and the reaction mixture stirred for a further 5minutes at about -60° C. The cooling bath was then removed and thereaction mixture allowed to warm to room temperature before beingtreated with water (230 ml). The organic phase was separated, combinedwith dichloromethane extracts (2×250 ml) of the aqueous phase, washedwith brine (500 ml), dried (MgSO₄) and evaporated under reducedpressure. The crude product was purified by distillation under reducedpressure to provide the title compound (6.8 g) as a clear oil, b.p.58-64° C. at 1.33 kPa (10 mm Hg). m/e 114.1 (M+H)⁺.

The somewhat unstable product was used immediately in the next step ofthe reaction sequence.

PREPARATION 22 N-N-(N-Methyl-3-piperidyl)-O-t-butyl-(S)-α-aspartyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compounds of Preparation 17 and Preparation 21by analogy with Preparation 19. Rf (diastereoisomers) 0.30 and 0.40 (SS10). Found: C,59.32; H,9.01; N,11.06. C₃₇ H₆₆ N₆ O₈ ; 0.40 CH₂ Cl₂requires C,59.35; H,8.90; N, 11.10%. m/e 723.9 (M+H)⁺.

PREPARATION 23 N- N-(2-Norbornyl)-O-t-butyl-(S)-α-aspartyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 17 and(+)-2-norbornanone by analogy with Preparation 19. Rf 0.65 (SS 10). m/e720.5 (M+H)⁺.

PREPARATION 24 N-(N-Cyclohexyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 17 and cyclohexanone byanalogy with Preparation 19. Rf 0.80 (SS 7). m/e 708 (M+H)⁺.

PREPARATION 25 N-(N-Cyclohexyl-O-t-butyl-(S)-α-aspartyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 18 and cyclohexanone byanalogy with Preparation 19. Rf 0.70 (SS 10). m/e 708.7 (M+H)⁺.

PREPARATION 26 N-N-(4-Tetrahydropyranyl)-O-t-butyl-(S)-α-aspartyl!-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 18 and4-tetrahydropyranone by analogy with Preparation 19. Rf 0.60 (SS 10).m/e 710.5 (M+H)⁺.

PREPARATION 27 N-N-(3,3-Dimethylcyclohexyl)-O-t-butyl-(S)-α-aspartyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 17 and3,3-dimethylcyclohexanone (J. Org. Chem., 1976, 41, 1069) by analogywith Preparation 19. Rf 0.62 (SS 10). m/e 736.3 (M+H)⁺.

PREPARATION 28 N-N-(3,3-Dimethylcyclohexyl)-O-t-butyl-(S)-α-aspartyl!-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 18 and3,3-dimethylcyclohexanone (J. Org. Chem., 1976, 41, 1069) by analogywith Preparation 19. Rf 0.62 (SS 10). m/e 736.9 (M+H)⁺.

PREPARATION 29 Cis-3,5-Dimethylcyclohexanone

A solution of 3,5-dimethyl-2-cyclohexenone (2.0 g, 16.1 mmol) in2-propanol (32 ml) was hydrogenated over 5% palladium on charcoal (200mg) at room temperature and 103 kPa (14.7 psi) for 4 hours, then dilutedwith 1:1 hexane:ether (150 ml) and filtered using a filter aid. Thefiltrate was evaporated under reduced pressure to provided thesubstantially stereochemically pure title compound (1.56 g) as a yellowoil. ¹³ C NMR spectroscopy revealed a cis:trans ratio of 150:3.

PREPARATION 30 N-N-(cis-3,5-Dimethylcyclohexyl)-O-t-butyl-(S)-α-aspartyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Two diastereoisomers were obtained from the title compounds ofPreparation 17 and Preparation 29 by analogy with Preparation 19 andseparated by chromatography on silica gel, using an elution gradient ofethyl acetate:hexane (3:7 to 4:6), in a ca. 4:1 ratio.

Diasteroisomer A (major isomer)

Rf 0.55 (SS 2). m/e 736.7 (M+H)⁺.

Diasteroisomer B (minor isomer)

Rf 0.34 (SS 2). m/e 736.9 (M+H)⁺.

PREPARATION 31 N-(N-Cycloheptyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 17 and cycloheptanone byanalogy with Preparation 19. Rf 0.56 (SS 10). m/e 722.9 (M+H)⁺.

PREPARATION 32 N-(N-Cycloheptyl-O-t-butyl-(S)-α-aspartyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 18 and cycloheptanone byanalogy with Preparation 19. Rf 0.57 (SS 10). m/e 722.5 (M+H)⁺.

PREPARATION 33 N-(N-Cyclopentyl-O-t-butyl-(S)-α-aspartyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 18 and cyclopentanone byanalogy with Preparation 19. Rf 0.56 (SS 10). m/e 694.5 (M+H)⁺.

PREPARATION 34 N-(N-Cyclooctyl-O-t-butyl-(S)-α-aspartyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 18 and cyclooctanone byanalogy with Preparation 19. Rf 0.58 (SS 10). m/e 737 (M+H)⁺.

PREPARATION 35 N-N-(2-Hydroxycyclohexyl)-O-t-butyl-(S)-α-aspartyl!-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

A solution of the title compound of Preparation 18 in tetrahydrofuran(0.2 M; 20 μl), a solution of 2-hydroxycyclohexanone in tetrahydrofuran(0.2M; 22 μl), a suspension of sodium triacetoxyborohydride intetrahydrofuran (0.2M; 30 μl) and a solution of glacial acetic acid intetrahydrofuran (0.2M; 20 μl) were combined and the resulting mixtureshaken at room temperature for 3 days, during which the solventevaporated. The crude product was purified by chromatography on silicagel, using dichloromethane:methanol (95:5) as eluant, to give the titlecompound. Rf 0.26 (SS 11). m/e 724.4 (M+H)⁺.

PREPARATION 36 N-N-(3-Tetrahydrothiopyranyl-O-t-butyl-(S)-α-aspartyl!-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 18 and3-tetrahydrothiopyranone by analogy with Preparation 35, but using anelution gradient of dichloromethane:methanol (100:0 to 95:5) forchromatographic purification. Rf 0.38 (SS 1 1). m/e 726.4 (M+H)⁺.

PREPARATION 37 N-N-(4-Tetrahydrothiopyranyl-O-t-butyl-(S)-α-aspartyl!-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

A solution of the title compound of Preparation 18 in 1,2-dichloroethane(0.2M; 25 μl), a solution of 4-tetrahydrothiopyranone in1,2-dichloroethane (0.2 M; 50 μl), a suspension of sodiumtriacetoxyborohydride in 1,2-dichloroethane (0.2 M; 50 μl) and 5 beadsof pre-activated (ca. 120° C. in vacuo) 4Å molecular sieves werecombined and the resulting mixture shaken at room temperature for 3days, during which the solvent evaporated. The crude product waspurified by chromatography on silica gel, using an elution gradient ofdichloromethane:methanol (100:0 to 95:5), to provide the title compound.Rf 0.79 (SS 10). m/e 726.3 (M+H)⁺.

PREPARATION 38N-(N-Cyclohexylmethyl)-O-t-butyl-(S)-α-aspartyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Sodium triacetoxyborohydride (185 mg, 0.88 mmol) was added in oneportion to a stirred mixture of the title compound of Preparation 18(276 mg, 0.44 mmol) and cyclohexanecarboxaldehyde (0.107 ml, 0.88 mmol)in dry tetrahydrofuran (5 ml) under nitrogen at room temperature, thenthe resulting mixture stirred for 3.5 hours and evaporated under reducedpressure. The residue was partitioned between ethyl acetate andsaturated aqeuous sodium bicarbonate solution, then the organic phaseseparated, washed with brine, dried (MgSO₄) and evaporated under reducedpressure to give a foam which was purified by chromatography on silicagel, using hexane:ethyl acetate (1:1) as eluant, to furnish the titlecompound (137 mg) as a crystallised foam. Rf 0.80 (SS 2). m/e 723(M+H)⁺.

PREPARATION 39 N-(N-Cyclopentylmethyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 17 andcyclopentanecarboxaldehyde (Org. Syn. Coll. Vol. 5, 1973, 320) byanalogy with Preparation 38, but using 1.5 mol. equivs. of sodiumtriacetoxyborohydride. Rf 0.27 (SS 2). α!_(D) ²⁵ -0.7° (c=0.7, CH₃ OH).m/e 708 (M+H)⁺.

PREPARATION 40N-(N-Cyclohexyl-N-methyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Aqueous formaldehyde solution (37%; 0.34 ml, 4.15 mmol) was added to astirred solution of the title compound of Preparation 24 (735 mg, 1.04mmol) in dichloromethane (15 ml) followed, 1 hour later, by sodiumtriacetoxyborohydride (440 mg, 2.08 mmol). After a further 1 hour, thereaction mixture was diluted with dichloromethane, washed sequentiallywith saturated aqueous sodium bicarbonate solution and brine, dried (Na₂SO₄) and evaporated under reduced pressure to give a gum (810 mg) whichwas purified by chromatography on silica gel, using an elution gradientof dichloromethane:methanol:0.880 aqueous ammonia (393:7:1 to 293:7:1),to afford the title compound (720 mg) as a white foam. Rf 0.32 (SS 12).Found: C,62.53; H,9.34; N,9.31. C₃₈ H₆₇ N₅ O; 0.50 H₂ O requiresC,62.44; H,9.37; N,9.58%. m/e 722 (M+H)⁺.

PREPARATION 41N-(N-Cyclohexyl-N-methyl-O-t-butyl-(S)-α-aspartyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 25 by analogy withPreparation 40. Rf 0.75 (SS 10). m/e 722.6 (M+H)⁺.

PREPARATION 42 N-N-Methyl-N-(3,3-dimethylcyclohexyl)-O-t-butyl-(S)-α-astartyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 27 by analogy withPreparation 40. Rf 0.78 (SS 10). m/e 751 (M+H)⁺.

PREPARATION 43N-(N-Cycloheptyl-N-methyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 31 by analogy withPreparation 40. Rf 0.74 (SS 10). m/e 736.6 (M+H)⁺.

PREPARATION 44N-(N-Cycloheptyl-N-methyl-O-t-butyl-(S)-α-aspartyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 32 by analogy withPreparation 40. Rf 0.74 (SS 10). m/e 736.7 (M+H)⁺.

PREPARATION 45 N-N-Methyl-N-(cis-3,5-dimethylcyclohexyl)-O-t-butyl-(S)-α-aspartyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from diastereoisomer A of the title compound of Preparation 30by analogy with Preparation 40. Rf 0.73 (SS 2). Found: C,63.49; H,9.45;N,9.24. C₄₀ H₇₁ N₅ O₈ ; 0.50 H₂ O requires C,63.30; H,9.56; N,9.23. m/e750.7 (M+H)⁺.

PREPARATION 46 N-N-Methyl-N-(3-methyl-3-cyclohexenyl)-O-t-butyl-(S)-α-aspartyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 20 by analogy withPreparation 40. Rf 0.75 (SS 2). m/e 734.5 (M+H)⁺.

PREPARATION 47N-(N-Cyclopentyl-N-methyl-O-t-butyl-(S)-α-aspartyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 33 by analogy withPreparation 40. Rf 0.76 (SS 10). m/e 708.5 (M+H)⁺.

PREPARATION 48N-(N-Cyclohexyl-N-ethyl-O-t-butyl-(S)-α-aspartyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

A solution of acetaldehyde in tetrahydrofuran (1M; 0.97 ml, 0.97 mmol)was added to a stirred solution of the title compound of Preparation 25(328 mg, 0.463 mmol) followed, 0.5 hour later, by sodiumtriacetoxyborohydride (216 mg, 1.02 mmol). After 18 hours, furtherportions of the acetaldehyde solution (1M; 0.34 ml, 0.34 mmol) andsodium triacetoxyborohydride (72 mg, 0.34 mmol) were added, then thereaction mixture stirred for 3 hours more and evaporated under reducedpressure. The residual solid was partitioned between ethyl acetate andwater, then the organic phase separated, washed sequentially withsaturated aqueous sodium bicarbonate solution and brine, dried (MgSO₄)and evaporated under reduced pressure. The crude product was purified bychromatography on silica gel, using hexane:ethyl acetate (1:1) aseluant, to provide the title compound (193 mg) as a white foam. Rf 0.87(SS 13). Found: C,62.42; H,9.36; N,9.13. C₃₉ H₆₉ N₅ O₈ ; 0.20 CH₂ Cl₂requires C,62.53; H,9.29; N,9.30%. m/e 736.3 (M+H)⁺.

PREPARATION 49 N-(2(S)-t-Butoxycarbonylmethyl)piperidinoacetyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

An aqueous solution of glutaraldehyde (50 wt. %; 0.1 ml, 0.50 mmol) wasadded to a stirred solution of the title compound of Preparation 17 (212mg, 0.338 mmol) in dichloromethane (5 ml) followed, 0.5 hour later, bysodium triacetoxyborohydride (109 mg, 0.51 mmol). After a further 1.75hours, the reaction mixture was diluted with dichloromethane (30 ml),then washed sequentially with saturated aqueous sodium bicarbonatesolution and brine, dried (MgSO₄) and evaporated under reduced pressureto give an oil which was purified by chromatography on silica gel, usinghexane:ethyl acetate (6:4) as eluant, to afford the title compound (137mg) as a glass-like solid. Rf 0.53 (SS 2). α!_(D) ²⁵ -11.70 (c=0.3, CH₃OH). m/e 694 (M+H)⁺.

PREPARATION 50 N-N-(9-Fluorenylmethoxycarbonyl)-O-t-butyl-(S)-α-glutamyl!-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

A suspension of 1-ethyl-3-(3-dimethylamino-1-propyl)carbodiimidehydrochloride (348 mg, 2.00 mmol) in dichloromethane (7 ml) was added inone portion to a stirred, ice-cooled mixture of the title compound ofPreparation 14 (470 mg, 1.03 mmol),N-fluorenylmethoxycarbonyl-(S)-glutamic acid-γ-t-butyl ester (426 mg,1.00 mmol), 1-hydroxybenzotriazole monohydrate (137 mg, 1.01 mmol),N-methylmorpholine (220 μl, 2.00 mmol) and dichloromethane (17 ml) undernitrogen. After 20 minutes the cooling bath was removed and the reactionmixture stirred for a further 17 hours and then evaporated under reducedpressure. The residue was partitioned between ethyl acetate and water,then the organic phase separated, washed with saturated aqueous sodiumbicarbonate solution and brine, dried (MgSO₄) and evaporated underreduced pressure to give an oil which was purified by columnchromatography on silica gel, using an elution gradient of hexane:ethylacetate (6:4 to 4:6), to afford the title compound (244 mg) as a foam.Rf 0.32 (SS 2). Found: C,65.40; H,7.93; N,7.66. C₄₇ H₆₇ N₅ O₁₀ requiresC,65.48; H,7.83; N,8.12%. m/e 640 (M-Fmoc+H)⁺.

PREPARATION 51 N-(O-t-Butyl-(S)-α-glutamyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 50 by analogy withPreparation 17. Rf 0.26 (SS 8). m/e 640 (M+H)⁺.

PREPARATION 52 N-(N-Cyclohexyl-O-t-butyl-(S)-α-glutamyl)-2(R,S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 51 and cyclohexanone byanalogy with Preparation 19. Rf 0.55 (SS 13). m/e 722 (M+H)⁺.

PREPARATION 53 N-t-Butoxycarbonyl-2(R,S)-(2-hydroxyethyl)piperidine

A solution of di-t-butyl dicarbonate (10.91 g, 50 mmol) in ethyl acetate(15 ml) was added to a stirred, ice-cooled solution of2(R,S)-(2-hydroxyethyl)piperidine (5.06 g, 50 mmol) in ethyl acetate (25ml), then the cooling bath was removed. After a further 1.5 hours thereaction mixture was evaporated under reduced pressure and the residuepurified by chromatography on silica gel, using hexane:ether (1:1) aseluant, to provide the title compound (8.46 g, 74%) as a clear oil. Rf0.25 (SS 14).

PREPARATION 54 N-t-Butoxycarbonyl-2(R,S)-2-(4-cyanophenoxy)ethyl!piperidine

Diethyl azodicaboxylate (1.73 ml, 11 mmol) was added to a stirred,ice-cooled solution of the title compound of Preparation 53 (2.29g, 10mmol) and 4-cyanophenol (1.31 g, 11 mmol) in tetrahydrofuran (75 ml),then the cooling bath was removed. After a further 18 hours the reactionmixture was evaporated under reduced pressure and the residue dissolvedin ether. The resulting solution was washed with 1M aqueous sodiumhydroxide solution and brine, dried (Na₂ SO₄) and evaporated underreduced pressure to give crude product which was purified bychromatography on silica gel, using hexane:ether (1:1) as eluant, tofurnish the title compound (2.86 g) as a clear oil which solidified onstanding. Rf 0.30 (SS 14). Found: C,69.20; H,8.32; N,8.42. C₁₉ H₂₆ N₂ O₃requires C,69.06; H,7.93; N,8.49%.

PREPARATION 55 2(R,S)- 2-(4-Cyanophenoxy)ethyl!piperidine

Trifluoroacetic acid (10 ml) was added to a stirred, ice-cooled solutionof the title compound of Preparation 54 (2.83 g, 8.56 mmol) and anisole(1.88 ml, 17.3 mmol) in dry dichloromethane (15 ml). After a further 1hour the reaction mixture was evaporated under reduced pressure, theresidue dissolved in water and the solution washed with ether, basifiedwith 2M aqueous sodium hydroxide solution and thrice extracted withether. The combined extracts were washed with brine, dried (Na₂ SO₄) andevaporated under reduced pressure to afford the title compound (1.43 g)as an oil which solidified on standing. Rf 0.15 (SS 7). m/e 231 (M+H)⁺.

PREPARATION 56 N-(N-t-Butoxycarbonyl-O-benzyl-(S)-α-aspartyl)-2(R,S)-2-(4-cyanophenoxyethyl!piperidine

Obtained as a white foam from the title compound of Preparation 55 andN-t-butoxycarbonyl-(S)-aspartic acid β-benzyl ester (1.2 mol. equiv.) byanalogy with Preparation 15, using hexane:ether (1:3) as the eluant forchromatographic purification. Rf (diastereoisomers) 0.30 and 0.41 (SS15). Found: C,66.88; H,6.75; N,7.80. C₃₀ H₃₇ N₃ O₆ requires C,67.27;H,6.96; N,7.84%. m/e 536 (M+H)⁺.

PREPARATION 57N-(O-Benzyl-(S)-α-aspartyl)-2(R,S)-{2-(4-cyanophenoxy)ethyl}piperidine

Trifluoroacetic acid (8 ml) was added to a stirred, ice-cooled solutionof the title compound of Preparation 56 (3.0 g, 5.6 mmol) and anistle(1.2 ml, 11.2 mmol) in dry dichloromethane (15 ml). After a further 2hours the reaction mixture was evaporated under reduced pressure, theresidue dissolved in ether and the solution extracted with water. Afterfive extractions a heavy oil separated and this was removed with theaqueous phase; a further twenty aqueous extractions were required toobtain all the desired product. The combined aqueous extracts (ca. 600ml), together with the insoluble oil, were basified to pH 11 with 10Maqueous sodium hydroxide solution and then extracted withdichloromethane. The organic extract was washed with brine, dried (Na₂SO₄) and evaporated under reduced pressure to provide the title compound(2.25 g) as a viscous oil. Rf (diastereoisomers) 0.28 and 0.38 (SS 7).m/e 436 (M+H)⁺.

PREPARATION 58 N-(N-Cyclohexyl-O-benzyl-(S)-α-aspartyl)-2(R,S)-2-(4-cyanophenoxy)ethyl!piperidine

Sodium triacetoxyborohydride (585 mg, 2.76 mmol) was added to a stirredsolution of the title compound of Preparation 57 (802 mg, 1.84 mmol),cyclohexanone (0.23 ml, 2.2 mmol) and glacial acetic acid (0.12 ml, 2mmol) in tetrahydrofuran (10 ml). After a further 1.7 hours the reactionmixture was evaporated under reduced pressure, the residue partitionedbetween ethyl acetate and saturated aqueous sodium bicarbonate solution,and the organic solution separated, washed with brine, dried (Na₂ SO₄)and evaporated under reduced pressure to give a gum (970 mg) which waspurified by chromatography on silica gel, using hexane:ethyl acetate(1:1) as eluant, to furnish the title compound (840 mg) as a gum. Rf0.25 (SS 2). Found: C,70.77; H,7.36; N,7.78. C₃₁ H₃₉ N₃ O₄ ; 0.50 H₂ Orequires C,70.70; H,7.65; N,7.98%. m/e 518 (M+H)⁺.

PREPARATION 59 N-(N-Cyclohexyl-O-ethyl/benzyl(9/1)-(S)-α-aspartyl)-2(R,S)- 2-(4-amidinophenoxy)ethyl!piperidinedihydrochloride

A stirred, ice-cooled solution of the title comopund of Preparation 58(810 mg, 1.56 mmol) in absolute ethanol (10 ml) which had been driedover 3Å molecular sieves was saturated with hydrogen chloride and thenleft at about 0° C. for 3 days. The reaction mixture was evaporatedunder reduced pressure, residual hydrogen chloride removedazeotropically with ethanol and the residue dried in vacuo to give theintermediate imino ether hydrochloride as a white foam. Rf 0.50 (SS 7).

A solution of this intermediate in ethanolic ammonia (1.96M; 5.2 ml,10.17 mmol) was heated at 50° C. for 3 hours and then evaporated underreduced pressure. The residue was dissolved in water, then the solutionwashed with ether, basified with 1M aqueous sodium hydroxide solutionand extracted with dichloromethane. The organic extract was washed withbrine, dried (Na₂ SO₄), treated with excess ethereal hydrogen chlorideand evaporated under reduced pressure to afford the title compound (790mg) as a white powder. Rf 0.30 (SS 16). Found: C,56.17; H,7.56; N,9.68.C₂₆ H₄₀ N₄ O₄ ; 2HCl; 0.125 CH₂ Cl₂ requires C,56.45; H,7.66; N,10.08%.m/e 473 (M+H)⁺ -ethyl ester.

The ratio of ethyl to benzyl ester (ca. 9:1) was indicated by ¹ H NMRspectroscopy.

PREPARATION 60 N-N-(N-Methyl-4-piperidyl)-O-benzyl-(S)-α-aspartyl!-2(R,S)-2-(4-cyanophenoxy)ethyl!piperidine

Obtained as a gum from the title compound of Preparation 57 andN-methyl-4-piperidone by analogy with Preparation 58. Rf(diastereoisomers) 0.28 and 0.33 (SS 7). m/e 533 (M+H)⁺.

PREPARATION 61 N-N-(N-Methyl-4-piperidyl)-O-ethyl/benzyl(2/3)-(S)-aspartyl!-2(R,S)-2-(4-amidinophenoxy)ethyl!piperidine trihydrochloride

Obtained as a white powder from the title compound of Preparation 60 byanalogy with Preparation 59. Rf (diastereoisomers) 0.17 and 0.24 (SS16). m/e 488 (M+H)⁺ -ethyl ester, 550 (M+H)⁺ -benzyl ester.

PREPARATION 62 N-t-Butoxycarbonyl-4-piperidone

A solution of di-t-butyl dicarbonate (56.8 g, 0.26 mole) in acetonitrile(100 ml) was added dropwise to a stirred, ice-cooled suspension of4-piperidone hydrochloride monohydrate (40.0 g, 0.26 mole) intriethylamine (26.3 g, 0.26 mole) and acetonitrile (300 ml). After afurther 5 days the reaction mixture was filtered and the filtrateevaporated under reduced pressure to give an off-white solid (69 g)which was purified by chromatography on silica gel, using hexane:ether(1:1) as eluant, to provide the title compound (37.5 g) as a whitesolid, m.p. 74-75° C. Rf 0.60 (SS 17). Found: C,60.36; H,8.78; N,6.88.C₁₀ H₁₇ NO₃ requires C,60.28; H,8.60; N,7.03%.

PREPARATION 63 N-N-(N-t-Butoxycarbonyl-4-piperidyl)-O-benzyl-(S)-α-aspartyl!-2(R,S)-2-(4-cyanophenoxy)ethyl!piperidine

Obtained as a gum from the title compounds of Preparation 57 andPreparation 62 by analogy with Preparation 58. Rf (diastereoisomers)0.33 and 0.43 (SS 18). Found: C,67.71; H,7.96; N,8.64. C₃₅ H₄₆ N₄ O₆requires C,67.94; H,7.49; N,9.05%. m/e 619 (M+H)⁺, 519 (M-Boc+2H)⁺.

PREPARATION 64 N- N-(4-Piperidyl)-O-benzyl-(S)-α-aspartyl!-2(R,S)-2-(4-cyanophenoxy)ethyl!piperidine

Obtained as a foam from the title compound of Preparation 63 by analogywith Preparation 55. Rf (diastereoisomers) 0.12 and 0.16 (SS 7). m/e 519(M+H)⁺.

PREPARATION 65 N-N-Methyl-N-(N-methyl-4-piperidyl)-O-benzyl-(S)-α-aspartyl!-2(R,S)-2-(4-cyanophenoxy)ethyl!piperidine

Obtained as a gum from the title compound of Preparation 64 by analogywith Preparation 40, but using 5 mol. equiv. of formaldehyde. Rf 0.60(SS 7). Found: C,70.23; H,8.17; N,9.84. C₃₂ H₄₂ N₄ O₄ requires C,70.30;H,7.74; N,10.25%. m/e 547 (M+H)⁺.

PREPARATION 66 N-N-Methyl-N-(N-methyl-4-piperidyl)-O-ethyl/benzyl(1/4)-(S)-α-aspartyl!-2(R,S)-2-(4-amidinophenoxy)ethyl!piperidine trihydrochloride

Obtained as a white powder from the title compound of Preparation 65 byanalogy with Preparation 59. Rf (diastereoisomers) 0.26 and 0.32 (SS16). m/e 502 (M+H)⁺ -ethyl ester, 564 (M+H)⁺ -benzyl ester.

PREPARATION 67 N-(N-4-Cycloheptenyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 17 and 4-cycloheptenone.(J. Org Chem., 1982, 42, 693) by analogy with Preparation 19. Rf 0.39(SS 2); 0.76 (SS 10). Found: C,63.04; H,9.12; N,9.65. C₃₅ H₆₅ N₅ O₈requires C,63.39; H,9.10; N,9.73%. m/e 720 (M+H)⁺.

PREPARATION 68N-(N-4-Cycloheptenyl-N-methyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 67 by analogy withPreparation 40. Rf 0.63 (SS 2); 0.66 (SS 8). Found: C,63.1 1; H,9.24;N,9.38. C₃₉ H₆₇ N₅ O₈ ; 0.50 H₂ O requires C,63.05; H,9.22; N,9.43%. m/e734 (M+H)⁺.

PREPARATION 69 N-(N-Cyclopropylmethyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 17 andcyclopropanecarboxaldehyde by analogy with Preparation 38. Rf 0.10 (SS2). α!_(D) ²⁵ -2.6° (c=0.6, CH₃ OH). m/e 681 (M+H)⁺.

PREPARATION 70 N-(N-3-Pentyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 17 and 3-pentanone byanalogy with Preparation 19. m/e 696.2 (M+H)⁺.

PREPARATION 71 N-(N-2-Cyclohexenyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Potassium carbonate (190 mg, 1.37 mmol) was added to a stirred solutionof the title compound of Preparation 17 (300 mg, 0.48 mmol) inacetonitrile (3 ml), followed by a solution of 3-bromocyclohexene (80mg, 0.50 mmol) in acetonitrile (1 ml), and the mixture heated underreflux for 24 hours. A further portion of 3-bromocyclohexene (80 mg,0.50 mmol) was then added to the cool reaction mixture and stirringunder reflux continued for a further 24 hours. The resulting reactionmixture was evaporated under reduced pressure and the residuepartitioned between ethyl acetate (150 ml) and water (150 ml). Theorganic phase was separated, washed with brine (150 ml), dried (MgSO₄)and evaporated under reduced pressure to give a white foam which waspurified by chromatography on silica gel, using hexane:ethyl acetate(2:1) as eluant, to provide the title compound (300 mg) as a white foam.Rf 0.43 (SS 2). Found: C,64.49; H,10.03; N,9.60. C₃₇ H₆₃ N₅ O₈ ; 0.67 C₆H₁₄ requires C,64.50; H,9.56; N,9.14%. m/e 706.3 (M+H)⁺.

PREPARATION 72N-(N-2-Cyclohexenyl-N-methyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 71 by analogy withPreparation 40. Rf 0.31 (SS 2). Found: C,63.33; H,9.17; N,9.26. C₃₈ H₆₅N₅ O₈ ; 0.07 CH₂ Cl₂ requires C,62.99; H,9.05; N,9.65%. m/e 720.3(M+H)⁺.

PREPARATION 73N-(N-3(R)-Methylcyclohexyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained as a mixture of diastereoisomers from the title compound ofPreparation 17 and 3(R)-methylcyclohexanone by analogy with Preparation19. Rf 0.47 and 0.58 (SS 2). Found: C,63.06; H,9.37; N,9.47. C₃₈ H₆₇ N₅O₈ requires C,63.21; H,9.35; N,9.70%. m/e 722.7 (M+H)⁺.

Repeated chromatography on silica gel, using hexane:ethyl acetate (3:1)as eluant, allowed isolation of the major, higher-running (Rf 0.58, SS2) diastereoisomer. m/e 722.7 (M+H)⁺.

PREPARATION 74N-(N-Methyl-N-3(R)-methylcyclohexyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained as a mixture of diastereoisomers from the title compound ofPreparation 73 (mixture of diastereoisomers) by analogy with Preparation40. Rf 0.90 (SS 19).

A single diastereoisomer of the title compound was also obtained byeffecting the corresponding N-methylation of the major, higher-runningdiastereoisomer from Preparation 73. Rf 0.57 (SS 2). Found: C,64.20;H,9.81; N,9.22. C₃₉ H₆₉ N₅ O₈ requires C,63.64; H,9.45; N,9.52%.

PREPARATION 75 N-N-Methyl-N-(2-norbornyl)-d-t-butyl-(S)-α-aspartyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 23 by analogy withPreparation 40. Rf 0.70 (SS 2). Found: C,63.23; H,9.18; N,9.41. C₃₉ H₆₇N₅ O₈ ; 0.05 CH₂ Cl₂ requires C,63.53; H,9.16; N,9.49%.

PREPARATION 76 3-Tetrahydropyranone

Obtained as a yellow oil from 3-hydroxytetrahydropyran (J. Org. Chem.,1970, 35, 898) by a procedure similar to that described for Preparation21.

PREPARATION 77 N-N-(3-Tetrahydropyranyl)-O-t-butyl-(S)-α-aspartyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compounds of Preparation 17 and Preparation 76by analogy with Preparation 19. Rf 0.79 (SS 8). Found: C,59.89; H,8.84;N,9.46. C₃₆ H₆₃ N₅ O₉ ; 0.20 CH₂ Cl₂ requires C,59.81; H,8.79; N,9.65%.

PREPARATION 78 N-N-Methyl-N-(3-tetrahydropyranyl)-O-t-butyl-(S)-α-aspartyl!-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 77 by analogy withPreparation 40. Rf 0.83 (SS 10). Found: C,60.14; H,9.01; N,9.21. C₃₇ H₆₅N₅ O₉ ; 0.20 CH₂ Cl₂ requires C,60.30; H,8.90; N,9.45%. m/e 723.8(M+H)⁺.

PREPARATION 79 3-Cyclohexenone

Obtained from anisole by analogy with the method described in J. Org.Chem., 1977, 42, 1051 for the preparation of 3-methyl-3-cyclohexenone(see Preparation 20) from 3-methylanisole. B.p. 66-69° C. 1.33 kPa (10mm Hg). ν c=o 1725 cm⁻¹. Found: C,73.45; H,8.54. C₆ H₈ O; 0.03 CH₂ Cl₂requires C,73.40; H,8.23%.

PREPARATION 80 N-(N-3-Cyclohexenyl-O-t-butyl-(S)-α-aspartyl)-2(S)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compounds of Preparation 17 and Preparation 79by analogy with Preparation 19. Rf 0.92 (SS 10). Found: C,61.56; H,8.79;N,9.18. C₃₇ H₆₃ N₅ O₈ ; 0.30 CH₂ Cl₂ requires C,61.25; H,8.76; N,9.57%.m/e 706.7 (M+H)⁺.

PREPARATION 81N-(N-3-Cyclohexenyl-N-methyl-O-t-butyl-(S)-α-aspartyl)-2(s)-{2-N-(N,N'-di-t-butoxycarbonylamidino)-4-piperidyloxy!ethyl}piperidine

Obtained from the title compound of Preparation 80 by analogy withPreparation 40. Rf 0.84 (SS 8). Found: C,62.80; H,9.15; N,9.31. C₃₈ H₆₅N₅ O₈ ; 0.10 CH₂ Cl₂ requires C,62.82; H,9.02; N,9.61%. m/e 720.6(M+H)⁺.

Biological activity

The following Table illustrates the in vitro inhibitory activitiesagainst thrombin and trypsin for a range of the compounds of theinvention.

                  TABLE    ______________________________________                 Ki(M)    EXAMPLE        THROMBIN  TRYPSIN    ______________________________________    3              4.1 × 10.sup.-9                             1.2 × 10.sup.-6    16             1.3 × 10.sup.-9                             7.2 × 10.sup.-7    17             3.5 × 10.sup.-8                             1.1 × 10.sup.-5    25             2.0 × 10.sup.-9                             3.6 × 10.sup.-7    29             1.6 × 10.sup.-8                             5.0 × 10.sup.-6    31             4.5 × 10.sup.-8                             1.3 × 10.sup.-6    ______________________________________

Safety profile

Certain compounds of the invention have been tested at 1 mg/kg i.v. inrat without showing any sign of adverse toxicity. One of these compoundshas also been tested at 10 mg/kg i.v. in rat and at doses of up to 10mg/kg i.v. in mouse; again no sign of adverse toxicity was apparent.

We claim:
 1. A compound of formula (I): ##STR25## or a pharmaceuticallyacceptable salt thereof, or a pharmaceutially acceptable solvate ofeither entity,wherein A is optionally monounsaturated C₄ -C₅ alkyleneoptionally substituted with C₁ -C₄ alkyl; B is C₁ -C₃ alkyleneoptionally substituted with C₁ -C₄ alkyl; R¹ is N-amidino-4-piperidyl or4-amidinophenyl; R² is C₄ -C₁₂ alkyl; (C₃ -C₈ cycloalkyl)C₁ -C₄alkylene; optionally methylene-bridged C₅ -C₈ cycloalkyl optionallysubstituted with one to three C₁ -C₄ alkyl groups or with hydroxy; C₅-C₈ alkenyl; C₅ -C₈ cycloalkenyl optionally substituted with C₁ -C₄alkyl; piperidyl N-substituted with C₁ -C₄ alkyl; tetrahydrothiopyranylor tetrahydropyranyl; and R³ is H or C₁ -C₄ alkyl optionally substitutedwith C₁ -C₄ alkoxy or with hydroxy; or R² and R³, together with thenitrogen atom to which they are attached, form a piperidine ring whichis optionally substituted with C₁ -C₄ alkyl or is optionallybenzo-fused.
 2. A compound according to claim 1 wherein the preferredstereoisomer is of formula (IA): ##STR26## .
 3. A compound according toclaim 2 wherein A is butylene; B is C₁ -C₂ alkylene; R² is C₄ -C₆ alkyl;(C₃ -C₆ cycloalkyl)CH₂ ; C₅ -C₈ cycloalkyl optionally substituted withone to three methyl groups or with hydroxy; norbornyl; C₆ -C₇cycloalkenyl optionally substituted with methyl; piperidyl N-substitutedwith methyl; tetrahydrothiopyranyl or tetrahydropyranyl; R³ is H, methylor ethyl; or R² and R³, together with the nitrogen atom to which theyare attached, form a piperidine ring substituted with methyl.
 4. Acompound according to claim 3 wherein A is butylene; B is methylene; R¹is N-amidino-4-piperidyl; R² is 3-pentyl; (C₅ -C₆ cycloalkyl)CH₂ ;cyclopentyl; cyclohexyl optionally substituted with one or two methylgroups; 2-hydroxycyclohexyl; 2-norbornyl; cycloheptyl; cyclooctyl;cyclohexenyl optionally substituted with methyl; cycloheptenyl;3-tetrahydrothiopyranyl; 4-tetrahydrothiopyranyl or 3-tetrahydropyranyl;and R³ is H, methyl or ethyl.
 5. A compound according to claim 4 whereinA is butylene; B is methylene; R¹ is N-amidino-4-piperidyl; R² iscyclohexyl; 3-methylcyclohexyl; 3,3-dimethylcyclohexyl;3,5-dimethylcyclohexyl; 2-norbornyl; cycloheptyl; cyclooctyl;3-cyclohexenyl; 3-methyl-3-cyclohexenyl or 4-cycloheptenyl; and R³ is Hor methyl.
 6. A compound according to claim 5 which is selectedfromN-(N-cycloheptyl-N-methyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine;N-(N-4-cycloheptenyl-N-methyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine;N-(N-methyl-N-3(R)-methylcyclohexyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine; andN-(N-cyclohexyl-N-methyl-(S)-α-aspartyl)-2(S)-2-(N-amidino-4-piperidyloxy)ethyl!piperidine;and pharmaceuticallyacceptable salts thereof, and pharmaceutically acceptable solvates ofeither entity.
 7. A pharmaceutical composition comprising a compound ofclaim 1, or a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable solvate of either entity, together with apharmaceutically acceptable diluent or carrier.
 8. A compound of formula(II): ##STR27## wherein R⁴ is C₁ -C₄ alkyl or benzyl, A, B, R² and R³are as previously defined in claim 1, and the amidino group in R¹ asdefined in claim 1 is optionally protected.
 9. A method of treating amammal to cure or prevent deep vein thrombosis (DVT) after surgery,major medical illness, paralysis, malignancy, prolonged immobilizationtrauma, application of lower limb plaster casts, or fractures of thelower limbs or pelvis; recurrent DVT; DVT during pregnancy when there isa previous history thereof; reocculsion following thrombolytic therapy;chronic arterial obstruction; peripheral vascular disease; acutemyocardial infarction; unstable angina; atrial fibrillation; thromboticstroke; transient ischaemic attacks; disseminated intravascularcoagulation; coagulation in extra-corporeal circuits; occulsion ofarterio-venous shunts and blood vessel grafts (including coronary arteryby-pass grafts); restonosis and occlusion following angioplasty;neurodegenerative disorders; inflammatory disorders; or scarring; whichcomprises treating said mammal with an effective amount of compound ofclaim 1, or a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable solvate of either entity.